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Effect of the N-terminal hydrophobic sequence of hepatitis B virus surface antigen on the folding and assembly of hybrid beta-galactosidase in Escherichia coli
Abstract
To investigate the mechanism of inclusion body formation and the effect of a hydrophobic sequence on the in vivo polypeptide folding, the aggregation caused by recombinant fusion beta-galactosidase in Escherichia coli was examined. Two plasmids were constructed: pTBG(H-) carried only the preS2 sequence of the hepatitis B virus surface antigen (HBsAg) in front of the beta-galactosidase gene (lacZ) while pTBG(H+) carried an additional sequence encoding the amino-terminal hydrophobic sequence of the S region of HBsAg between preS2 and lacZ. Unlike cells expressing the fusion protein not containing the hydrophobic sequence, E. coli JM109/pTBG(H+) exhibited temperature-sensitive production of beta-galactosidase. As the culture temperature increased the activity decreased dramatically. This decrease in activity was not due to a decrease in fusion polypeptide production, but rather the fusion polypeptides containing the hydrophobic sequence aggregated within the cells at high temperature. However once the fusion polypeptides folded into proper conformation at low temperature, they maintained the activity even at high temperature. The results indicate that aggregation is a consequence of incorrect folding and assembly of the polypeptides, and is not derived from the native structure. The aggregates of the pTBG(H+)-encoded fusion polypeptides did not revert to active form when the culture temperature was lowered.
... PreS2-b-galactosidase is a tetrameric fusion protein composed of identical ,120 kDa protomers consisting of the preS2 domain of the hepatitis B surface antigen (HBsAg) fused to the N-terminus of E. coli b-galactosidase (Lee et al., 1990). Whereas this protein is almost completely soluble in E. coli at temperatures as high as 428C, the closely related fusion protein preS2-S H -bgalactosidase aggregates extensively at all temperatures owing to the presence of a 29-amino-acid hydrophobic insertion, corresponding to the S H domain of the HBsAg between the preS2 and b-galactosidase regions (Lee et al., 1990). ...
... PreS2-b-galactosidase is a tetrameric fusion protein composed of identical ,120 kDa protomers consisting of the preS2 domain of the hepatitis B surface antigen (HBsAg) fused to the N-terminus of E. coli b-galactosidase (Lee et al., 1990). Whereas this protein is almost completely soluble in E. coli at temperatures as high as 428C, the closely related fusion protein preS2-S H -bgalactosidase aggregates extensively at all temperatures owing to the presence of a 29-amino-acid hydrophobic insertion, corresponding to the S H domain of the HBsAg between the preS2 and b-galactosidase regions (Lee et al., 1990). We have previously demonstrated that both preS2-b-galactosidase and preS2-S H -b-galactosidase exhibit a strong requirement for DnaK±DnaJ±GrpE for proper folding, but have little or no need for the GroEL± GroES system (Thomas and Baneyx, 1996a,b). ...
... Both heteroligomeric and homodimeric forms of Rubisco have been shown to require the GroE chaperonins for proper folding in E. coli (Goloubinoff et al., 1989). The GroEL± GroES team has also been implicated in the folding of pBR322 derivative coding for the lacZ a-peptide under tac promoter control (Amp r ) (Lee et al., 1990) pTBG(H 2 ) pBR322 derivative coding for preS2-b-galactosidase under tac promoter control (Amp r ) (Lee et al., 1990) pTBG(H 1 ) pBR322 derivative coding for preS2-S H -b-galactosidase under tac promoter control (Amp r ) (Lee et al., 1990) pRR2119 pBR322 derivative encoding the gene for homodimeric ribulose bisphosphate carboxylase (Rubisco) from R. rubrum under lac promoter control (Amp r ) (Somerville and Somerville, 1984) pMal-c2 pBR322 derivative encoding a cytoplasmic fusion between the maltose binding protein and the lacZ a-peptide under tac promoter control. Also encodes lacI q (Amp r ) ...
DnaK–DnaJ–GrpE and GroEL–GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either ClpB or HtpG at 42°C leads to increased aggregation of preS2-β-galactosidase, a fusion protein whose folding depends on DnaK–DnaJ–GrpE, but not GroEL–GroES. However, only the ΔclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL–GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S′-β-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK–DnaJ, but not GroEL–GroES, restore efficient folding in ΔclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK–DnaJ–GrpE team to interact with newly synthesized polypeptides.
... Thus, redistribution of the α-fragment to the insoluble fraction in cells would be predicted to lead to a reduction in the level of β-gal activity. Three extant systems for monitoring protein misfolding in vivo rely on the ability of misfolding proteins to co-translationally induce improper folding of a C-terminally fused marker protein [40][41][42] . As such, slow misfolding or aggregation events may escape detection by these schemes. ...
... Previously reported systems for monitoring in vivo protein folding rely on coupling the misfolding of an N-terminal target to the misfolding of a C-terminal marker protein such as β-gal (ref. 40), green fluorescent protein (GFP) (ref. 41), or chloramphenicol acetyltransferase (CAT) 42 . ...
... 41), or chloramphenicol acetyltransferase (CAT) 42 . Therefore, these methods report on events that occur during the process of folding 40 , perhaps even cotranslationally 41 . It is unclear if these methods will detect the slow aggregation reactions that are hallmarks of many degenerative diseases. ...
Protein misfolding is the basis of a number of human diseases and presents an obstacle to the production of soluble recombinant proteins. We present a general method to assess the solubility and folding of proteins in vivo. The basis of this assay is structural complementation between the alpha- and omega- fragments of beta-galactosidase (beta-gal). Fusions of the alpha-fragment to the C terminus of target proteins with widely varying in vivo folding yield and/or solubility levels, including the Alzheimer's amyloid beta (A beta) peptide and a non-amyloidogenic mutant thereof, reveal an unambiguous correlation between beta-gal activity and the solubility/folding of the target. Thus, structural complementation provides a means of monitoring protein solubility/misfolding in vivo, and should find utility in the screening for compounds that influence the pathological consequences of these processes.
... In this work, we used the preS2-SЈ--galactosidase fusion protein as a model to find novel multicopy suppressors (3,35) of protein aggregation in vivo. Previous in vitro studies (17,32) have shown that this fusion protein, which is soluble at 30 and 37°C, is highly prone to aggregation at 42°C. It has also been shown that increased levels of DnaK and DnaJ and of the heat shock sigma factor 32 result in an increase in the recovery of active preS2-SЈ--galactosidase in crude bacterial extracts (17,33). ...
... Previous in vitro studies (17,32) have shown that this fusion protein, which is soluble at 30 and 37°C, is highly prone to aggregation at 42°C. It has also been shown that increased levels of DnaK and DnaJ and of the heat shock sigma factor 32 result in an increase in the recovery of active preS2-SЈ--galactosidase in crude bacterial extracts (17,33). We took advantage of this differential solubility of preS2-SЈ--galactosidase observed in vitro to develop a selection procedure for gene products that would prevent preS2-SЈ--galactosidase aggregation in vivo. ...
... E. coli strain JC196 (parental strain MG1655) was used as a host strain in all experiments since it carries the ⌬(lacZ)M15 deletion (absence of residual -galactosidase activity). Plasmid pTBGHϩ, which encodes the fusion preS2-SЈ--galactosidase, has been described previously (17). We plated strain JC196 carrying plasmid pTBGHϩ on MacConkey agar-1% lactose at 37 and 43°C in order to check the lactose-positive and -negative phenotypes. ...
We used preS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43 degrees C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-beta-galactosidase aggregation at 43 degrees C. Overexpression of PIMT reduces the amount of preS2-S'-beta-galactosidase found in inclusion bodies at 43 degrees C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-beta-galactosidase and increases its thermal stability in E. coli crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.
... GFP has been fused to the C-term of the target proteins and the fluorescence of the resulting fusion protein considered indicative of its correct folding [11] . Similarly , fusions with β-galactosidase [12], chloramphenicol acetyltransferase [13] or the structural complementation between the C-term fused α-fragment of the β-galactosidase with the ω-fragment [14] have been described. However , the systems relying on GFP have at least two strong limitations, namely the long lag-phase (95 min) necessary to the chromophore to form and the persistence of the fluorescence even after protein aggregation [15,16]. ...
... Probes for the aggregation detection have been proposed in the past11121314, but most of them were fused to the protein of interest and a direct interaction effect could not be ruled out. A non invasive probe was built by fusing the promoter element of the heat shock transcription factor σ 32 with the GFP and used to estimate the cell response to stress [22]. ...
The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the beta-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree.
The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The beta-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the beta-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system.
The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.
... Intracellularly, the low temperature enables a slow-rate synthesis of foreign proteins, so that the nascent peptide chains may be allowed more time to fold in a proper way. Meanwhile, the misfolded or aggregated proteins are more sensitive to the proteolytic enzymes under low cultivation temperature, which allows properly folded protein to be secreted and misfolded protein to be degraded (Lee et al., 1990;Li et al., 2001). Zhong et al. found that high temperature would prolong the accumulation process of the nascent proteins in the endoplasmic reticulum (ER), which led to the overload of ER and cell death eventually. ...
Regardless of bacteria or eukaryotic microorganism hosts, improving their ability to express heterologous proteins is always a goal worthy of elaborate study. In addition to traditional methods including intracellular synthesis process regulation and extracellular environment optimization, some special or extreme conditions can also be employed to create an enhancing effect on heterologous protein production. In this review, we summarize some extreme environmental factors used for the improvement of heterologous protein expression, including low temperature, hypoxia, microgravity and high osmolality. The applications of these strategies are elaborated with examples of well-documented studies. We also demonstrated the confirmed or hypothetical mechanisms of environment stress affecting the host behaviors. In addition, multi-omics techniques driving the stress-responsive research for construction of efficient microbial cell factories are also prospected at the end.
... To show the membrane localization of the identified peptides, we fused a hexahistidine tag to the C terminus of Arp1 and conducted an electron microscopic analysis of immunogold-labeled samples using a His tag antibody (Fig. 3A). The micrographs revealed large inclusion bodies in the cells, a common artifact when overexpressing hydrophobic peptides (32,33), which are strongly decorated with antibody-attached gold particles. Outside these aggregates the antibodies localized both in the cytoplasm as well as in the cell periphery, indicating that a major fraction of free Arp1 (outside the inclusion bodies) does populate the membrane. ...
De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.
... Moreover, higher temperatures may also lead to exposure of more hydrophobic surfaces during peptide folding and favour hydrophobic interaction and thus may predispose hTopoI to aggregation. Misfolded and aggregated proteins are more susceptible to the intracellular proteolytic degradation [22,23]. Therefore, inducing protein expression at a lower temperature may help to reduce protein misfolding and allow more properly folded protein to be secreted into the culture medium. ...
Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression.
Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions.
Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.
... Furthermore, TEM images of cells expressing mEGFP-free amphiphilic proteins with the corresponding hydrophilic versus hydrophobic ratio show that artificial organelle formation does not depend on the mEGFP-tag (Supplementary Table 1c). In addition, it is known that the orientation of hydrophilic or hydrophobic domains at the amino or carboxy terminus of proteins 23 influences the structural and functional properties of proteins. Confirming these findings we show that the translational order of the protein domains (hydrophobic or hydrophilic first) influences the self-assembly process and the structures formed ( Fig. 2 and Supplementary Fig. 7a). ...
Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.
... The resultant 1227-bp fragment of PCR product was purified and cloned into analogous sites in pYA3137, an Asd+ vector with a pUC18 origin of replication, to form pYA3201 (Fig. 1); pYA3201 expresses mZP3 aa 23 to 424 with 4 additional amino acids (Met, Pro, Glu, and Phe) added on the N-terminal because of the vector-specifying amino acids. Because the C-terminal end of mZP3 is highly hydrophobic, a characteristic that may negatively affect expression of the protein [24], 180 bp of cDNA encoding 60 amino acids at the C-terminal end were deleted by digestion with Ban II and HindIII. The 5' and 3' overhanging ends of the linearized plasmid were bluntended with T4 DNA polymerase and self-ligated to form pYA3202, which expresses a truncated mZP3 protein, aa 23 to 364. ...
Ovarian ZP3, the primary sperm receptor, is a major glycoprotein of mouse zona pellucida (ZP). Because antibodies raised against ZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. This study explored the possibility of using an attenuated Salmonella typhimurium vaccine strain expressing recombinant ZP3 to elicit an antibody response and infertility in mice. A cDNA sequence generated by the polymerase chain reaction encoding 342 amino acid residues (23-364) of the mouse (m)ZP3 was cloned into an Asd+ vector. An avirulent Salmonella vaccine strain stably expressed the ZP3 polypeptide and colonized the internal organs of mice after oral inoculation. Oral immunization of female BALB/c mice with the recombinant Salmonella vaccine strain expressing mZP3 induced significant levels of anti-native ZP IgG antibodies in serum and IgA antibodies in vaginal secretions. The IgG antibodies thus induced also bound to ZP in vivo. When mated with males, 3 of 6 females immunized with the recombinant Salmonella were infertile. In contrast, none of the mice that received Salmonella containing the vector plasmid produced antibodies to ZP and all were fertile. No ovarian inflammation was observed in the immunized mice at autopsy. The results suggest a potential oral contraceptive vaccine to control populations of rodent vectors of disease and to induce reversible infertility in humans.
... At present we do not understand the significance of this observation. Hybrid protein inclusion body formation may be related to the increasing hydrophobicity of the RsaA N-terminus, a factor known to influence inclusion body formation in other systems (Lee et al. 1990). In support of this idea, the region encompassing amino acids 36-69 is twice as rich in hydrophobic amino acids as the region of RsaA encompassing amino acids 2-35. ...
The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5'-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (delta CenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9%. No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell. RsaA:PhoA hybrid proteins were also exported from the E. coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA. Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.
... The resultant 1227-bp fragment of PCR product was purified and cloned into analogous sites in pYA3137, an Asd+ vector with a pUC18 origin of replication , to form pYA3201 (Fig. 1); pYA3201 expresses mZP3 aa 23 to 424 with 4 additional amino acids (Met, Pro, Glu, and Phe) added on the N-terminal because of the vector-specifying amino acids. Because the C-terminal end of mZP3 is highly hydrophobic, a characteristic that may negatively affect expression of the protein [24], 180 bp of cDNA encoding 60 amino acids at the C-terminal end were deleted by digestion with Ban II and HindIII. The 5' and 3' overhanging ends of the linearized plasmid were bluntended with T4 DNA polymerase and self-ligated to form pYA3202, which expresses a truncated mZP3 protein, aa 23 to 364. ...
Ovarian ZP3, the primary sperm receptor, is a major glycoprotein of mouse zona pellucida (ZP). Because antibodies raised against ZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. This study explored the possibility of using an attenuated Salmonella typhimurium vaccine strain expressing recombinant ZP3 to elicit an antibody response and infertility in mice. A cDNA sequence generated by the polymerase chain reaction encoding 342 amino acid residues (23-364) of the mouse (m)ZP3 was cloned into an Asd+ vector. An avirulent Salmonella vaccine strain stably expressed the ZP3 polypeptide and colonized the internal organs of mice after oral inoculation. Oral immunization of female BALB/c mice with the recombinant Salmonella vaccine strain expressing mZP3 induced significant levels of anti-native ZP IgG antibodies in serum and IgA antibodies in vaginal secretions. The IgG antibodies thus induced also bound to ZP in vivo. When mated with males, 3 of 6 females immunized with the recombinant Salmonella were infertile. In contrast, none of the mice that received Salmonella containing the vector plasmid produced antibodies to ZP and all were fertile. No ovarian inflammation was observed in the immunized mice at autopsy. The results suggest a potential oral contraceptive vaccine to control populations of rodent vectors of disease and to induce reversible infertility in humans.
... Strain or plasmid Genotype or description Source or reference Strains MC4100 araD139 D(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR Lab collection JGT3 MC4100 DclpB::kan [39] JGT20 MC4100 dnaK756 thr::Tn10 [39] JGT32 MC4100 dnaK756 thr::Tn10 DclpB::kan [39] JGT6 MC4100 zjd::Tn10 groES30 [39] JGT38 MC4100 zjd::Tn10 groES30 DclpB::kan [39] Plasmids pMM103 pACYC184 derivative encoding the trc promoter, a multiple cloning site and the rrnB terminator (Chl r ) [32] pClpB95/80 pMM103 derivative encoding clpB95/clpB80 under trc transcriptional control (Chl r ) [32] pClpB95 pMM103 derivative encoding clpB95 under trc transcriptional control (Chl r ) [32] pClpB80 pMM103 derivative encoding clpB80 under trc transcriptional control (Chl r ) [32] pSR22 pBR322 derivative in which the [47] Abbreviations are: Amp, ampicillin; Chl, chloramphenicol; Spc, spectinomycin; r superscript, resistant. I-Ting Chow et al. / FEBS Letters 579 (2005) 4242–4248site (MGSSHHHHHHSSGLVPRGSH). ...
ClpB/Hsp104 collaborates with the Hsp70 system to promote the solubilization and reactivation of proteins that misfold and aggregate following heat shock. In Escherichia coli and other eubacteria, two ClpB isoforms (ClpB95 and ClpB80) that differ by the presence or absence of a highly mobile 149-residues long N-terminus domain are synthesized from the same transcript. Whether and how the N-domain contributes to ClpB chaperone activity remains controversial. Here, we show that, whereas fusion of a 20-residues long hexahistidine extension to the N-terminus of ClpB95 interferes with its in vivo and in vitro activity, the same tag has no detectable effect on ClpB80 function. In addition, ClpB95 is more effective than ClpB80 at restoring the folding of the model protein preS2-beta-galactosidase as stress severity increases, and is superior to ClpB80 in improving the high temperature growth and low temperature recovery of dnaK756 DeltaclpB cells. Our results are consistent with a model in which the N-domain of ClpB95 maximizes substrate processing under conditions where the cellular supply of free DnaK-DnaJ is limiting.
Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-α mutant (cIFN) were
serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that
could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture
of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only
one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on
SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro
conversion assay based on incubation with different concentrations of β-mercaptoethanol. The results showed that only a wound
such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were
broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers.
Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete
formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which
result in sharp decrease of bioactivity of cIFN in bioreactor.
The extracellular lipase Lip2 from Yarrowia lipolytica (YlLip2) has been widely used as a catalyst in industry for biodiesel production, esters synthesis and optical resolution of chiral compounds. YlLip2 was successfully expressed in Pichia pastoris under the control of the AOX1 promoter. To further improve lipase production in P. pastoris, we constructed multi-copy lipase gene Lip2 clones using high Zeocin concentration screening. Two clones which can resist 1000 µg/mL Zeocin showed higher lipase production in shaking flasks. Southern blot analysis showed that three lipase genes were integrated into the genome of P. pastoris GS115. Low temperature (25°C) could also improve the extracellular lipase yield. A high lipase activity of 42 900 U/mL (3.4 g total protein and 2.16 g lipase per liter) was obtained using the three copy integration clone in fed-batch fermentation using basal salt medium, which was 2.5-fold higher than that using a single copy clone. The high yield of lipase YlLip2 in P. pastoris could decrease the price of this biocatalyst and accelerate its industrial applications.
We used preS2-S'-beta-galactosidase, a three domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli to search for multicopy suppressors of protein aggregation and inclusion bodies formation, and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C by selecting pink lactose-positive colonies on a background of white lactose-negative colonies after transformation of bacteria with an E. coli gene bank. We found only two multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C, protein isoaspartate methyltransferase (PIMT) and the membrane components ChbBC of the N,N'-diacetylchitobiose phosphotransferase transporter. We have previously shown that PIMT overexpression reduces the level of isoaspartate in preS2-S'-beta-galactosidase, increases its thermal stability and consequently helps in its solubilization at 43 degrees C (Kern et al., J. Bacteriol. 187, 1377-1383). In the present work, we show that ChbBC overexpression targets a fraction of preS2-S'-beta-galactosidase to the membrane, and decreases its amount in inclusion bodies, which results in its decreased thermodenaturation and in a lactose-positive phenotype at 43 degrees C. Cross-linking experiments show that the inner membrane protein ChbC interacts with preS2-S'-beta-galactosidase. Our results suggest that membrane docking of aggregation-prone proteins might be a useful method for their solubilization.
Over-expression of recombinant proteins in microbial hosts results in the formation of active soluble protein or of insoluble aggregates (inclusion bodies). Efficient in vitro refolding strategies have been developed to reactivate inactive proteins from inclusion bodies. Co-expression of molecular chaperones may provide a tool to promote correct structure formation of recombinant proteins in vivo.
Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.
Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.
The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).
PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.
We have systematically investigated the influence of mutations in the sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines on the folding of preS2-beta-galactosidase. This 120 kDa fusion protein between the hepatitis B surface antigen preS2 sequence and beta-galactosidase was synthesized in a highly soluble and enzymatically active form in wild-type Escherichia coli cells cultured at temperatures between 30 degrees C and 42 degrees C, but aggregated extensively in an rpoH165 (Am) mutant. Proper folding was partially restored upon co-overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced. The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40-60% lower relative to a dnaK756 mutant or isogenic wild-type cells at 30 degrees C and 37 degrees C. At 42 degrees C, only 10-40% of the wild-type activity was present in each of the early-folding-factor mutants. Although the synthesis levels of preS2-beta-galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37 degrees C or 42 degrees C. By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2-beta-galactosidase at temperatures up to 42 degrees C. Our results are discussed in terms of the current understanding of the E. coli protein-folding cascade. The potential usefulness of heat-shock protein mutants for the production of soluble proteins in an inclusion-body form is addressed.
A transcriptional gene fusion between the cspA promoter and the lacZ gene was constructed to assess the usefulness of cold shock promoters for low-temperature protein expression. Synthesis of beta-galactosidase was efficiently repressed at 37 degrees C but rapidly induced upon transfer to the 15-to-30 degrees C range, leading to a three- to fivefold increase in specific activity relative to control cultures. Although the initial rates of beta-galactosidase accumulation at 20 degrees C were twice those measured at 15 degrees C, prolonged incubation at 20 degrees C, but not 15 degrees C, led to a dilution of activity due to repression of the promoter and cell division.
The aggregation-prone fusion protein preS2-S'-beta-galactosidase was used as a model system to compare the efficiencies of the IPTG-inducible tac promoter and the low-temperature-inducible cspA promoter in directing the expression of soluble recombinant polypeptides at reduced growth temperatures in Escherichia coli. At 37 degrees C, the fusion protein was produced at high levels from the tac promoter, but aggregated quantitatively in a biologically inactive form. In contrast, little preS2-S'-beta-galactosidase was synthesized from the cspA promoter at this temperature, presumably due to transcript instability. The highest yields of active enzyme were obtained following temperature downshift from 37 to 30 degrees C for the tac promoter and 25 degrees C for the cspA promoter. At 25 degrees C, the kinetics of accumulation of beta-galactosidase activity, ratios of soluble to insoluble fusion protein, and synthesis rates of preS2-S'-beta-galactosidase were virtually identical for both promoters for a period of 2 h postinduction. Thereafter, the cspA promoter became repressed, whereas synthesis of the fusion protein continued with the tac system. Following transfer to 10 degrees C, the tac promoter was almost completely inhibited while the cspA promoter was able to direct the synthesis of soluble preS2-S'-beta-galactosidase for up to 2 h. However, the levels of active enzyme produced were approximately threefold lower than those measured at 25 degrees C. Overexpression of native CspA had no effect on the accumulation of active preS2-S'-beta-galactosidase from the cspA promoter. It is therefore unlikely that CspA acts as it own positive inducer. Our results indicate that the cspA promoter can efficiently substitute for the tac system at 25 degrees C and may be particularly valuable for the expression of highly aggregation-prone or unstable gene products at 10 degrees C.
The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished. Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'-beta-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.
Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-ICL) were constructed and expressed in Saccharomyces cerevisiae cells. We observed subcellular localization of expressed products of the mutant CT-ICL genes by immunoelectron microscopy. An unknown structure termed a protein aggregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa et al. (1996) J. Electron Microsc. 45: 491–497).
We chose two typical cells harbouring the mutant ICL genes Δ550 and Δ237–339 to analyse the ultrastructure and three-dimensional (3D) structure of PABs. The PABs had a homogeneous matth with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PAB5 could not be separated from the cytoplasm or mitochondria under a confocal fluorescence microscope, 3D reconstruction of serial electron micrographs clearly showed the PAB was an independent structure of varying size and had the shape of an incomplete sphere. A cell was sometimes observed to have multiple PABs.
We recently identified RrmJ (alias FtsJ), the first encoded protein of the rrmJ-hflB heat shock operon, as an Um(2552) methyltransferase of the 23S rRNA. We now report that the rrmJ-deficient strain exhibits growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S(30) rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity. Polysome profile analysis of the rrmJ strain reveals an increase in the proportion of free 30S and 50S subunits at both 30 and 42 degrees C. These results suggest that the RrmJ-catalyzed methylation of Um(2552) in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures.
DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES. However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.
Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.
Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.
Human interferon–2 (IFN–2), an 18 kD, acidic protein, human interferon– (IFN–), a 17 kD, basic protein, and the interferon–induced murine protein Mx (72 kD) were all found in aggregates when produced in genetically engineered strains of E. coli grown at 37°C. However, at a growth temperature of 23–30°C, 30–90% of the recombinant protein was soluble. The temperature effect was not directly dependent on the concentration of the protein and was observed for several E. coli strains and for different plasmid constructions. Lysates of non–transformed E. coli grown at either temperature rendered initially soluble human recombinant IFN–2 insoluble at 37° but not at 0° or 30°C. Insolubilization was not abolished by nuclease treatment, and may involve sulfhydryl group shuffling, as sulfhydryl reducing agents added to a mock lysate gave a similar temperature dependent precipitation.
A strong promoter has been cloned on a series of plasmid vectors that facilitate the expression of cloned genes. This promoter, named tac [first described by DeBoer et al. (inRodriguez, R.L. and Chamberlin, M.J. (Eds.),Promoters, Structure and Function. Praeger, New York, 1982, pp. 462–481)] contains the -10 region of the IacUV5 promoter and the −35 region of the trp promoter. Our vectors contain various cloning sites followed by transcription termination signals. In addition, we describe plasmids that facilitate the conversion of the lac promoter to the stronger tac promoter. Thus, preexisting gene fusions using the lac or the lacUV5 promoter can be readily converted to tac promoter gene fusions without changing the ribosome-binding site (RBS). The tac promoter is repressed in lacIQ strains and can be induced by isopropylthio-β-d-galactoside (IPTG). Studies of expression of the cI repressor of bacteriophage λ show that the tac promoter is at least five times more efficient than the lacUV5 promoter. Under optimal conditions λ repressor constitutes up to 30% of the total cellular protein.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
We have used the chloramphenicol acetyltransferase (cat) gene expression system to study the effect of the X protein of hepatitis B virus (HBV) on viral enhancers. Plasmids containing the HBV enhancer and the core gene promoter linked to the cat gene were cotransfected with a plasmid containing the X gene into the human hepatoma cell line PLC/PRF/5. Our results indicate that the transfected X gene caused a trans-activation of the HBV enhancer. If a frameshift mutation or a deletion in the X structural gene was created, this trans-activation function was abolished. This result and the observation that the frameshift mutation did not alter the transcription of X mRNA suggest that the X protein is the trans-activating factor. Using similar techniques, we found that the X protein was also capable of trans-activating the simian virus 40 (SV40) and Rous sarcoma virus enhancers (pSV2cat and pRSVcat) in CV-1 cells. However, trans-activation of the SV40 enhancer by the X protein was not observed in COS-1 cells. By cotransfecting pSV2cat and the X gene with a plasmid containing either the intact SV40 genome, the SV40 genome devoid of the T-antigen (T-ag) gene, or only the T-ag gene, we demonstrated that SV40 T-ag can suppress trans-activation by the X protein. SV40 T-ag did not inhibit expression of the X gene or inactivate the X protein. The most probable mechanism of this inhibition is that T-ag competes with the X protein for a common target.
The in vivo accumulation of polypeptide chains in the form of aggregated non-native states is a problem in many applications of biotechnology. In the maturation pathway of the thermostable P22 tailspike endorhamnosidase, the folding and chain association intermediates can be distinguished from the native tailspikes in crude extracts of phage-infected Salmonella cells. Temperature-sensitive folding mutations, at many sites in the chain, destabilize these conformational intermediates preventing the formation of native tailspikes at restrictive temperatures (Goldenberg, D. P., Smith, D. H., and King, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7060-7064). We report here that both wild type and mutant tailspike polypeptide chains which fail to reach the native state accumulate in an aggregated state. These off-pathway aggregates form from a thermolabile intermediate in the productive folding pathway. These aggregation reactions are suppressed by lowering the temperature of maturation. Similar off-pathway steps from folding intermediates may account for the non-native aggregates often found in the expression of cloned genes in heterologous hosts.
To express the bacterio-opsin (bop) gene in Escherichia coli, we have employed the inducible expression vectors pIN-II-A, -B, and -C (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The vectors contain three cloning sites early in the E. coli lipoprotein gene (lpp) which is transcribed from tandem lpp and lac promoters. The bop gene was modified so as to delete the N-terminal leader sequence and then cloned into each of the three cloning sites to encode three different lipoprotein/bacterio-opsin fusions. Expression of the fusions was demonstrated both in vitro and in vivo. The fusion protein was estimated to be about 0.05% of the total cell protein. The cause for the low level of expression apparently was neither an inadequate level of mRNA nor degradation of the protein. However, expression of the fusions caused inhibition of the growth of the host to varying extents. One fusion protein was purified from E. coli membranes to homogeneity by immunoaffinity chromatography followed by preparative gel electrophoresis. The purified fusion protein generated a bacteriorhodopsin-like chromophore on treatment with defined lipid/detergent mixtures and retinal. When reconstituted into vesicles, the protein pumped protons on illumination comparably to the reconstituted native bacterio-opsin.
Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.
We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.
Temperature-sensitive mutations in the gene for the thermostable tail spike of phage P22 interyFere with the folding and subunit association pathway at the restrictive temperature but not with the activity or stability of the protein once matured. The local sites of these mutations and the mutant amino acid substitutions have been determined by DNA sequencing. Of 11 temperature-sensitive folding mutations, 3 were replacements of glycine residues by polar residues, and three were replacements of threonine residues by residues unable to form a side-chain H-bond. There were no proline replacements. Two of the temperature-sensitive sites in which threonine residues were replaced by isoleucine residues were homologous. These sequences probably maintain the correct local folding pathway at higher temperatures. The temperature-sensitive amino acid substitutions appear to destabilize a thermolabile intermediate in the wild-type folding pathway or to increase the rate of a competing off-pathway reaction.
Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.
The sequence of hepatitis B virus DNA contains an open reading frame which codes for a not-yet-identified protein of at least 389 amino acids. Only the products starting at the third (GP33/GP36) or the fourth (P24/GP27) initiation signal have been characterized as components of the viral surface antigen. We found a larger protein, P39, and its glycosylated form, GP42, in hepatitis B virus particles and viral surface antigen filaments. Immunological cross-reactions showed that P39/GP42 is partially homologous to P24/GP27 and GP33/GP36. The unique portion of its sequence bound monoclonal antibodies which had been induced by immunization with hepatitis B virus particles. Proteolytic cleavage patterns and subtype-specific size differences suggested that the sequence of P39 starts with the first initiation signal of the open reading frame. Its amino-terminal part (pre-s coded) is exposed at the viral surface and, probably, is highly immunogenic. A model is presented of how the open reading frame for the viral envelope leads to defined amounts of three different proteins.
The minor glycoproteins from hepatitis B surface antigen, GP33 and GP36, contain at their carboxy-terminal part the sequence of the major protein P24. They have 55 additional amino acids at the amino-terminal part which are coded by the pre-S region of the viral DNA.
To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.
The cornerstone of the new biotechnology industry is the ability to construct organisms which, when cultured, will produce large quantities of desired proteins. Each chapter of Maximising Gene Expression discusses a particular question related to the overall series of events which leads to obtaining large amounts of protein. Topics include the control of plasmid replication and stable inheritance in E. Coli and yeast; the initiation of mRNA synthesis; the determinants of mRNA stability; the control of the translation initiation process; what is known about protein degradation. Contents: E. Coli promoters. Yeast promoters. Protein coding genes of higher eukarayotes. The instability of messenger RNA in bacteria. Replication control of the ColE1-type plasmids. Copy number and stability of yeast plasmids. Translational initiation. Biased codon usage. The selective degradation of abnormal proteins in bacteria. Detection of proteins produced by recombinant DNA techniques. Mechanism and practice.
Denatured pro-subtilisin accumulated as aggregates in the Escherichia coli periplasmic space when pro-subtilisin was fused to the OmpA signal peptide using a high expression secretion vector, pIN-III-ompA, and only a small fraction of pro-subtilisin was properly folded to produce active subtilisin. Here we report a substantial improvement in the control of folding and a 16-fold increase of active subtilisin production by inducing the expression of the pre(ompA)-pro-subtilisin gene with a very low inducer concentration (0.005 mM IPTG) at a low culture temperature (23°C). These results indicate that the rate of protein synthesis and the culture temperature are important factors for proper folding of proteins secreted across the cytoplasmic membrane using pIN-III-ompA as vector.
Hepatitis B virus (HBV) is the cause of a debilitating and potentially fatal disease, and possibly also of primary hepato-cellular carcinoma1. A vaccine against hepatitis B would therefore be of considerable biomedical significance. One such vaccine, produced from the 22-nm particle form of hepatitis B surface antigen (HBsAg), has recently been found effective in clinical tests2, but being derived from the sera of chronic carriers, it is expensive and its supply limited. The application of recombinant DNA methods can, in principle, provide a limitless source of vaccine. HBV DNA has been cloned in bacteria and the genes coding for HBsAg and hepatitis B core antigen (HBcAg) identified3–5. Small amounts of both antigens have been synthesized in Escherichia coli
3,6,7 and to enhance this synthesis we have constructed plasmids capable of expressing the genes for the antigens under the control of the efficient tryptophan (trp) operon regulatory region8. We describe here the construction of such recombinant plasmids which direct the synthesis of high levels of HBcAg and a β-lactamase: HBsAg fusion polypeptide.
Optimal application of protein engineering technology will require understanding how the amino acid sequence of a polypeptide chain specifies its spatial conformation. It is now clear that polypeptide chain folding and subunit assembly proceeds through sequential pathways involving defined intermediates. The critical amino acid instructions directing these pathways appear to be dispersed through the sequence. Recent genetic approaches have begun to identify which residues in a chain are important in maintaining the pathway, and avoiding aggregated states. Investigations of the P22 tailspike endorhamnosidase suggest that one general class of mutants, tss mutants, may be particularly informative in identifying sequences determining the conformation of the intermediates. Certain inherited human diseases may represent single amino acid substitutions causing folding defects.
Unusually low culture temperature, such as 20C, was shown to be preferable for the synthesis of active human interferon- (IFN-) inE.
coli harboring a recombinant plasmid. TheE.
coli cells cultured at 20C gave 8.6-fold higher IFN- activity than those cultured at 37C. However, almost the equal amounts of IFN- protein were accumulated in both cells cultured at 20C and at temperature higher than 20C, suggesting that IFN- might exist as an active form in the cells cultured at 20C, while as a rather denatured form in the cells cultured at higher temperature.
A pBR322-derived expression vector, plasmid pKD1, was constructed containing the strong leftward promoter (pp) of bacteriophage λ the ribosome-binding site (RBS) of the ell gene of λ, and a unique downstream NdeI restriction site for construction of an ATG initiation codon. The section of the pol gene of Moloney murine leukemia virus (M-MLV) that codes for reverse transcriptase (RT) was cloned into the NdeI site of this vector generating the plasmid pRT103. Upon thermal induction, enzymatically active RT was expressed in Escherichia coli [pRT103]. The identity of this activity was confirmed by its template specificity and its sensitivity to inhibition by immunoglobulin G (IgG) prepared against authentic murine RT. RT represented 20% of the newly synthesized protein m these cells 20 min after induction.
The accumulation of newly synthesized polypeptide chains expressed from cloned genes as non native aggregates has become an important factor in the recovery of such proteins. Studies of both the refolding of denatured proteins in vitro, and of in vivo folding and maturation pathways, indicate that aggregates derive from specific partially folded intermediates and not from mature native, or fully unfolded proteins. The aggregation process in both homologous and heterologous cytoplasms may be driven by partial intracellular denaturation of intermediates, for example by high temperature, or by the absence of a critical factor—prosthetic group, sub-unit, chaperone—during the maturation process. All of these processes appear to be highly specific and subject to modification by genetic engineering of the intermediates, or alteration of their environment. This requires appreciation of the properties of such intermediates as distinct from the native states.
A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.
MOST proteins are denatured at temperatures above 50-60°C, although some enzymes, especially those from thermophilic organisms, remain active at temperatures up to 80-90 °C. The determination of the three-dimensional structure of the thermostable protease thermolysin showed that heat-stable proteins do not contain unusual structural features absent from less stable proteins1,2. Furthermore, the amino acid sequences of similar proteins from both mesophilic and thermophilic sources have been shown to be homologous, suggesting that the respective structures are similar3,4. Nevertheless, such homologous amino acid sequences also include many differences which obscure those amino acid changes actually responsible for differences in thermostability. We report here the structure of a temperature sensitive (ts) mutant of T4 phage lysozyme. This permits the first direct comparison of two protein structures in which all differences are directly related to a change in thermal stability. It is shown that, except for the replacement of a partially exposed arginine by a histidine, the three-dimensional structure of the ts lysozyme is virtually identical with that of native lysozyme.
DNA extracted from hepatitis B virus Dane particles has been cloned in bacteria using a plasmid vector. A full-length clone has been examined by restriction endonuclease analysis, and the nucleotide sequence of an 892-base pair fragment from cloned hepatitis B viral DNA encoding the surface antigen gene is reported. The amino acid sequence deduced from the DNA indicates that the surface antigens is a protein consisting of 226 amino acids and with a molecular weight of 25,398. The portion of the gene coding for this protein apparently contains no intervening sequences.
The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.
One of the open reading frames on hepatitis B virus (HBV) DNA comprises the coding region (designated the env gene) for the virus envelope proteins. Studies on messenger RNA transcription suggest that this gene has the potential to code for three related proteins: (1) a protein of 226 amino acids identified as a major protein constituent of the HBV envelope, termed S-protein; (2) a protein with 55 additional amino acids at the N-terminal coded for by a portion of the env gene upstream of the S-gene (pre-S); (3) a protein corresponding to the entire env gene (pre-S + S). Synthetic peptides from the N-terminals of proteins (2) and (3), and antisera to them have been used to study the occurrence and properties of pre-S sequences. The results presented here provide unambiguous evidence that all three env encoded proteins are present in HBV particles; synthetic peptides corresponding to the gene encoding pre-S are highly immunogenic and can be used in diagnostic tests for detection in human sera of antibodies preferentially recognizing HBV; such antibodies, specific for pre-S determinants, are elicited during hepatitis B infection and by immunization with HBV proteins (2) and (3); the hepatitis B vaccine licensed in the United States does not contain pre-S proteins; and the pre-S proteins of the HBV envelope contain domains specifically recognized by liver cells. These findings suggest that pre-S determinants are important in virus-neutralizing responses and should be present in HBV vaccines.
DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed.
Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral
particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free
system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of
its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals,
we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately
100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the
wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able
to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s
function to direct portions of the molecule across the membrane and are able to perform this function even when positioned
in an internal protein domain.
Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.
The S gene encoding the major surface polypeptide of hepatitis B virus is preceded by the region pre-S(2) with a capacity to code for 55 amino acid residues. In the product of region pre-S(2), the sequence of 19 amino acid residues (amino acids 14-32 from the N terminus) representing an area of high local hydrophilicity is shared by viral strains of subtypes adr, ayw, and ayr; residue 22, phenylalanine, is replaced by leucine in a strain of the other subtype, adw. A synthetic peptide vaccine involving these 19 amino acid residues, when given to two chimpanzees, raised antibodies that bound to viral particles and protected the animals from challenge with 10(6) chimpanzee infectious doses of hepatitis B virus.
Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Hepatitis B is a widespread viral disease. In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers. The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg). Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins. Recently, the HBV genome has been cloned in E.coli. Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported. We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1). Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen.
Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.
A series of hybrid genes has been constructed by fusion of the gene (lamB) coding for the outer membrane λ receptor protein (LamB), and the gene coding for the cytoplasmic enzyme β-galactosidase, lacZ. The resultant hybrid proteins of this series contain varying amounts of LamB at the NH2 terminus and a constant amount of functional β-galactosidase at the COOH terminus. The amount of lamB-coded protein present varies from two to about 270 amino acids of the 450 amino acids found in the mature LamB protein. The largest hybrid protein is novel, exhibiting several unique properties. It is efficiently localized to the outer membrane, exhibits an extremely low β-galactosidase specific activity, and is insoluble in Triton X-100. Other hybrid proteins, containing shorter LamB sequences, are localized less efficiently or not at all. The results suggest that additional information within LamB, residing downstream from the NH2-terminal signal sequence, is required for proper routing of the protein to the outer membrane.
A 809 bp Sau 3A – Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II – Hpa I and 712 bp Xba I – Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV⊟adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP
SS-39, and pTRP SS–50, respectively. The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant
expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody. Interestingly, the growth of transformants
harbouring pTRP SS-39 and pTRP SS–50 was not inhibited and these transformants expressed a considerable level of the HBsAg.
Minicells harbouring pTRP SS–6, pTRP SS–39, and pTRP SS–50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons,
respectively.
Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.
A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described. Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95%. The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.
About 200 million people are chronic carriers of hepatitis B surface antigen (HBsAg), but since hepatitis B virus (HBV) cannot be propagated in vitro, HBsAg transcription has been studied only in cell lines containing HBV DNA integrated into chromosomes, and HBsAg-related mRNAs 2.0 to 2.5 kilobases (kb) long have been described. We have analysed the transcripts produced in an infected chimpanzee liver and in a rat cell line containing HBV DNA. In contrast to previous suppositions we report here that the major S gene transcript initiates close to the S gene, that is, within the 'pre-S' region and is processed/polyadenylated at a site situated within the core gene. The efficiency of processing/polyadenylation at this site varies between the chimpanzee liver and the rat cell line studied. The S gene promoter does not contain a TATA box but instead has a sequence homologous to that which positions the 5' ends of the major simian virus 40 (SV40) late transcript.
Escherichia coli that has been genetically manipulated by recombinant DNA technology to synthesize human insulin polypeptides (A chain, B chain, or proinsulin) contains prominent cytoplasmic inclusion bodies. The amount of inclusion product within the cells corresponds to the quantity of chimeric protein formed by the bacteria. At peak production, the inclusion bodies may occupy as much as 20 percent of the Escherichia coli cellular volume.
- P Valenzuela
- P Gray
- M Quiroga
- J Zaldivar
- H M Goodman
- W Rutter
Valenzuela, P., Gray, P., Quiroga, M., Zaldivar, J., Goodman, H.
M. & Rutter, W. (1979) Nature 280, 815-819.