Protein Expression and Purification

Published by Elsevier
Online ISSN: 1096-0279
Print ISSN: 1046-5928
The importance of eggs as a source of specific antibodies is well recognized. Egg yolk contains 8--20mg immunoglobulins (IgY) per milliliter. However, the major problem in separating IgY is to remove the high concentrations of lipids in egg yolk. We first used water dilution method to get the supernatant containing IgY, then purified the antibody by caprylic acid-ammonium sulfate method, and obtained specific antibody with satisfactory purity and activity. By comparison of these several methods, each has its advantages, one can be chosen to purify IgY according to practical need. The purified IgY produced by the immunized chickens can stain the human peripheral blood mononuclear cell effectively when labeled with fluorescent FITC.
The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS-PAGE. Optimal pH and temperature were 5.0 and 40 degrees C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be (1)AGSYRSVAYFVDWAI(15). The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).
Trifluoromethyl ketones are potent inhibitors of a variety of serine hydrolases. Based on this chemistry improved affinity chromatography procedures were developed for juvenile hormone esterase from insects. New affinity gels were prepared by binding rationally designed ligands to epoxy-activated Sepharose. One ligand is 8-mercapto-1,1,1-trifluoro-2-octanone which has a methylene group replacing a sulfide sulfur beta to the carbonyl of the trifluoromethyl ketone of the previously reported ligand, 3-(4-mercaptobutylthio)-1,1,1-trifluoro-2-propanone. With many loading levels and esterases, the original gel bound enzymes too tightly, resulting in elution difficulties. This replacement of the sulfur beta to the ketone thought to interact with the catalytic serine decreases the binding capacity of the gel at similar loading by approximately 56% compared to the affinity gel with the thioether. However, elution of the enzyme from the column can be accomplished with less potent inhibitors such as 3-n-butylthio- or 3-n-pentylthio-1,1,1-trifluoro-2-propanone, which can easily be removed from the enzyme by dialysis in the presence of the detergent n-octyl beta-D-glucopyranoside. An alternative approach allowing elution with less potent inhibitors involved varying concentrations of the previous high-affinity ligand to optimize the concentration of ligand on the column. Low concentrations of the high-affinity ligand also allowed the use of less potent eluting agents. These two improved affinity chromatography systems have been successfully used to purify juvenile hormone esterase of Heliothis virescens to near homogeneity with a 30-90% recovery of recombinant esterase secreted into the cell media in a baculovirus expression system. The purity of the esterase after affinity chromatography with newly prepared gel was comparable to that produced using the original affinity system based on analyses by SDS-PAGE and isoelectric focusing. A library of affinity gels with ligands of different affinities used at several loading levels and a library of eluting inhibitors of varying potency facilitate the rational selection of conditions for the affinity purification of esterases.
SDS-PAGE gel (14%) of fractions from the purification of 2 0 aminobiphenyl-2,3-diol 1,2-dioxygenase from Pseudomonas sp. LD2, strain MB1332. Double banding of tryptic digest-indistinguishable species is indicated by an arrow. Lane 1, lysate; lane 2, centrifuged lysate; lane 3, pooled Q-Sepharose fractions; lane 4, ammonium sulfate cut; lane 5, pooled butyl-Sepharose fractions; and lane 6, pooled Sephacryl-200 active fractions. Lane 7, FMC molecular weight standards (5, 10, 15, 25, 35, 50, 75, 100, and 200 kDa are visible in gel).
SDS-PAGE gel (8-25%) of fractions from the purification of 2 0 aminobiphenyl-2,3-diol 1,2-dioxygenase from recombinant E. coli RR110. Lane 1, lysate; lane 2, centrifuged lysate; lane 3, pooled QSepharose fractions; lane 4, ammonium sulfate cut; lane 5, pooled butyl-Sepharose fractions; and lane 6, FMC molecular weight standards (5, 10, 15, 25, 35, 50, and 75 kDa are visible in gel).
Purification of 2 0 aminobiphenyl-2,3-diol 1,2-dioxygenase from 110 g wet cell paste Pseudomonas sp. LD2, strain MB1332
Carbazole is a nitrogen-containing heteroaromatic compound that occurs as a widespread and mutagenic environmental pollutant. The 2'aminobiphenyl-2,3-diol 1,2-dioxygenase involved in carbazole degradation was purified to near electrophoretic homogeneity from Pseudomonas sp. LD2 by a combination of ion-exchange chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. This purification was challenging due to the great instability of the enzyme under many standard conditions. The enzyme was also purified to electrophoretic homogeneity from recombinant Escherichia coli expressing the 2'aminobiphenyl-2,3-diol 1,2-dioxygenase-encoding gene cloned from Pseudomonas sp. LD2. The molecular mass of the native enzyme was determined by gel filtration to be 70 kDa. The subunit molecular masses were determined to be 25 and 8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase is an [alpha2beta2] heterotetramer. The optimal temperature and pH for the enzymatic production of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) from 2,3-dihydroxybiphenyl were determined to be 40 degrees C and 8.0, respectively. The maximum observed specific activity on 2,3-dihydroxybiphenyl was 48.1 mmol HOPDA min(-1) mg(-1). This indicated a maximum observed turnover rate of 360,000 molecules HOPDA enz(-1) s(-1). The K'm inhibition constant Ks and Vmax on 2,3 dihydroxybiphenyl were determined to be 5 microM, 37 microM, and 44 mmol min(-1) mg(-1), respectively. These results show that 2'aminobiphenyl-2,3-diol 1,2-dioxygenase is a meta-cleavage enzyme related to the 4,5-protocatechuate dioxygenase family, with comparable purification challenges posed by intrinsic enzyme instability.
The galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-alpha-D-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan-protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 micromol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galbeta1-3Gal and Galbeta1-4GlcNAc derivatives known as substrates of the beta1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galbeta1-3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galbeta1-3Gal.
An Escherichia coli recombinant system produced soluble and full-length beta-1,3-glucanase type II (BglII) cloned from the yeast-lytic actinomycete Oerskovia xanthineolytica. The expression system was designed to produce recombinant BglII with a six-histidine peptide fused to the carboxy end of the protein. The expression level was optimized to produce 30% of total protein of E. coli as the recombinant protein, releasing 75% to the extracellular space. The 43-kDa recombinant protein was purified by IMAC to homogeneity and its molecular and biochemical characteristics were studied, showing that there are no important functional differences with those properties described for the BglII purified from O. xanthineolytica.
There has been much recent interest in alpha-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for alpha-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 degrees C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.
A human alpha-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 micrograms/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG-Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed alpha-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal alpha-2,3-linked sialic acid among various acceptors. The apparent Km values of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.
To characterize an exo-beta-1,3-glucanase (ExgP) of an isolated fungal strain with high laminarin degradation activity, identified as Penicillium sp. KH10, heterologous secretory expression of the ExgP was performed in Aspergillus oryzae. Deduced amino acid sequence of the exgP gene possibly consisted of 989 amino acids which showed high sequence similarity to those of fungal exo-beta-1,3-glucanases belonging to the glycoside hydrolase (GH) family 55. Notably, the purified recombinant ExgP showed a single protein peak in the native state (by gel-permeation chromatographic analysis), but showed two protein bands in the denatured state (by SDS-polyacrylamide gel electrophoresis). These two polypeptides exhibited activity in a coexisting state even under reducing conditions, suggesting that non-covalent association of both polypeptides took place. Taken together with the nucleotide sequence information, the ExgP precursor (104kDa) would be proteolytically processed (cleaved) to generate two protein fragments (42 and 47kDa) and the processed products (polypeptide fragments) would be assembled each other by a non-covalent interaction. Moreover, one of the matured ExgP polypeptides was N-glycosylated by the post-translational modification.
An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.
The endo-beta-1,4-mannanase encoding gene man1 of Aspergillus aculeatus MRC11624 was amplified from mRNA by polymerase chain reaction using sequence-specific primers designed from the published sequence of man1 from A. aculeatus KSM510. The amplified fragment was cloned and expressed in Saccharomyces cerevisiae under the gene regulation of the alcohol dehydrogenase (ADH2(PT)) and phosphoglycerate kinase (PGK1(PT)) promoters and terminators, respectively. The man1 gene product was designated Man5A. Subsequently, the FUR1 gene of the recombinant yeast strains was disrupted to create autoselective strains: S. cerevisiae Man5ADH2 and S. cerevisiae Man5PGK1. The strains secreted 521 nkat/ml and 379 nkat/ml of active Man5A after 96 h of growth in a complex medium. These levels were equivalent to 118 and 86 mg/l of Man5A protein produced, respectively. The properties of the native and recombinant Man5A were investigated and found to be similar. The apparent molecular mass of the recombinant enzyme was 50 kDa compared to 45 kDa of the native enzyme due to glycosylation. The determined K(m) and V(max) values were 0.3 mg/ml and 82 micromol/min/mg for the recombinant and 0.15 mg/ml and 180 micromol/min/mg for the native Man5A, respectively. The maximum pH and thermal stability were observed within the range of pH 4-6 and 50 degrees C and below. The pH and temperature optima and stability were relatively similar for recombinant and native Man5A. Hydrolysis of an unbranched beta-1,4-linked mannan polymer released mannose, mannobiose, and mannotriose as the main products.
Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris. The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P. pastoris AOX1 promoter. Secretion yields up to 60 mg/liter were obtained in synthetic medium. The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A. niger with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the S. cerevisiae signal peptide was correctly processed in P. pastoris. The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5. Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P. pastoris and the absence of glycosylation. The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A. niger. Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C. The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes. P. pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form. A. niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P. pastoris.
Glycogen branching enzyme (GlgB, EC catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.
We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.
Inositol polyphosphates are the most widespread second messenger molecules in eukaryotic cells. Human Type I inositol 1,4,5-triphosphate (Ins(1,4,5)P(3)) 5-phosphatase removes the D-5 position phosphate from soluble Ins(1,4,5)P(3,) a key event in cell signaling particularly in Ca(2+) homeostasis. In this study, the cDNA encoding human Type I Ins(1,4,5)P(3) 5-phosphatase was subcloned into a modified pMAL expression vector. This plasmid produces a recombinant protein in fusion with affinity tags located at its N-terminus, consisting in a maltose binding protein (MPB) and an octa-histidine stretch. The construction was transformed into Escherichia coli BL21 (DE3) expression strain. This dual tag strategy allows the purification of milligrams of highly purified protein. The recombinant human Type I Ins(1,4,5)P(3) 5-phosphatase is active and can thus be used for functional and structural studies.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.
A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed. The plasmid directed abundant synthesis of the fusion protein in Escherichia coli. The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes. A procedure was developed to isolate the fusion protein in a relatively pure and soluble form. Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase. The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart.
Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 mannose and decorated with alpha-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the alpha-1,6 linked mannose backbone by the M-Pol I complex. Constructs encoding soluble portions of the M-Pol I subunits, Mnn9p and Van1p from Saccharomyces cerevisiae, were expressed in Pichia pastoris. Both subunits had to be expressed in the same strain to obtain the recombinant proteins. Recombinant M-Pol I was made only by the KM71 strain transformed with two vectors: one encoding Mnn9p and the other encoding Van1p. Soluble secreted M-Pol I was purified by sequential chromatography on DEAE-Trisacryl, GDP-Hexanolamine-Sepharose and Superdex 200. Characterization of the purified complex indicates that recombinant M-Pol 1 is a Mnn9p-Van1p heterodimer. Purified M-Pol I was active with alpha-1,6 mannobiose as acceptor and GDP-mannose as donor. HPLC identified five products confirmed to be 3-7 mannose residues long. Digestion with linkage-specific alpha-mannosidases revealed that the linkage formed is exclusively alpha-1,6. No alpha-1,2 mannosyltransferase activity, reported previously for M-Pol I immunoprecipitates from cell extracts was detected. These results provide further information on the role of M-Pol I in mannan biosynthesis.
Fructose-1,6-bisphosphatase is one of the key enzymes of the gluconeogenic pathway. It catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. Fructose-1,6-bisphosphatase from the extreme thermophilic bacterium Thermus thermophilus has been purified by crystallisation approach. The final well-shaped crystals have been obtained using vapour diffusion sitting-drops in the presence of PEG 400 as the precipitating agent. The initially obtained native twinned crystals diffracted up to 1.2Å resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 Å resolution and belong to the space groups I422 with cell dimensions (i) a=b=108.8Å, c=336.3Å showing two molecules in the asymmetric unit, and (ii) a=b=113.7Å, c=151.0Å with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9Å resolution. For further biochemical and biophysical investigations recombinant fructose-1,6-bisphosphatase has been produced in Escherichia coli. Both native (dissolved crystals) and recombinant material have been characterised by SDS-PAGE, N-terminal sequencing and MALDI-MS.
An important Calvin cycle enzyme, chloroplast fructose-1, 6-bisphosphatase (FBPase) from wheat, has been cloned and expressed up to 15% of the total cell protein using a pPLc expression vector in Escherichia coli by replacing the codons in the 5'-terminal encoding sequence with optimal and A/T-rich ones. The overexpressed wheat FBPase is soluble, fully active, and heat stable. It can be purified by chromatography in turn on DEAE-Sepharose and Sephacryl S-200, and around 15 mg of purified enzymes (>95%) is obtained from 1 liter of cultured bacteria. Its special activity is 8.8 u/mg, K(cat) is 22.9/S, K(m) is 121 microM, and V(max) is 128 micromol/min. mg. The recombinant FBPase can be activated by DTT, Na(+), or low concentrations of Li(+), Ca(2+), Zn(2+), GuHCl, and urea, while it can be inhibited by K(+) or NH(+)(4).
A procedure has been developed for high-level expression of Trypanosoma brucei fructose-bisphosphate aldolase in Escherichia coli. Therefore, a specific restriction site was introduced by mutagenesis at the front of the gene, enabling its ligation in an expression plasmid, immediately downstream of the regulatory sequences. Growth conditions were established for production of high amounts of soluble and active enzyme. Aldolase was purified to near-homogeneity from the soluble fraction of the bacterial lysate by nuclease treatment, differential precipitation steps, and passage over a CM-Sepharose column. From a 1-liter culture of E. coli cells, 60-120 mg of purified protein that is essentially indistinguishable in physicochemical and kinetic properties and in stability from the enzyme purified from trypanosomes grown in infected laboratory animals was reproducibly obtained.
The Class II fructose 1,6-bisphosphate aldolase (fda, Rv0363c) from the pathogen Mycobacterium tuberculosis H37RV was subcloned in the Escherichia coli vector pT7-7 and purified to near homogeneity. The specific activity (35 U/mg) is approximately 9 times higher than previously reported for the enzyme partially purified from the pathogen. Attempts to express the enzyme with an N-terminal fusion tag yielded inactive, mostly insoluble protein. The native recombinant enzyme is zinc-dependent and has a catalytic efficiency for fructose 1,6-bisphosphate cleavage higher than most Class II aldolases characterized to date. The aldolase has a Km of 20 microM, a kcat of 21 s(-1), and a pH optimum of 7.8. The molecular mass of the enzyme subunits as determined by mass spectrometry is in agreement with the mass calculated on the basis of its gene sequence minus the terminal methionine, 36,413 Da. The enzyme is a homotetramer and retains only two zinc ions per tetramer when transferred to a metal-free buffer, as determined by ICP-MS and by a colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) as a chelator. The E. coli expression system reported in this study will facilitate the further characterization of this enzyme and the screening for potential inhibitors.
Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms.
Full-length cDNA encoding pea cytoplasmic fructose-1,6-bisphosphatase (cyFBPase) was cloned from a pea cDNA library. The cloned cDNA was introduced into the Escherichia coli expression vector pET-15b. The recombinant cyFBPase was expressed in E. coli BL21 (DE3) cells in a soluble form and purified to homogeneity by Ni(+)-NTA affinity chromatography. The identity of the recombinant cyFBPase was confirmed by SDS-PAGE and immunoblot analysis using a polyclonal anti-His tag antibody. The recombinant cyFBPase was active at neutral pH ranges (6.6-9.0) and thermostable as other cyFBPases. The activation energy (E(a)) and Arrhenius frequency factor were 17.4 kcal/mol and 2.6 x 10(12)/s, respectively. The K(M) and V(max) values of the recombinant enzyme were calculated as 10.47 microM and 109 micromol/min, respectively. In case of removal of histidine tag, the K(M) value was calculated as 5.03 microM. The recombinant enzyme was non-competitively and competitively inhibited by AMP and fructose-2,6-bisphosphate, respectively.
Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.
The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.
Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere. This makes SBPase an appropriate target for intensive study. We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag. The identity of the recombinant SBPases was confirmed by SDS-PAGE analysis and immunological detection with a specific antibody. Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography. Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity. Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts. The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be one of the deadliest diseases in the world. TB resurged in the late 1980s and now kills more than 2 million people a year. Possible factors underlying the reemergence of TB are the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant (MDR) strains, patient noncompliance in completing the standard "short-course" therapy, and decline of health care systems. Thus, there is a need for the development of new antimycobacterial agents to treat MDR strains of M. tuberculosis, to provide for more effective treatment of latent tuberculosis infection, and to shorten the treatment course to improve patient compliance. The shikimate pathway is an attractive target for antimicrobial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent in mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of M. tuberculosis. The M. tuberculosis aroE-encoded shikimate dehydrogenase was PCR amplified, cloned, sequenced, and expressed in Escherichia coli BL21(DE3). Recombinant protein expression was achieved by a low-cost and simple protocol. Although cell lysis resulted in the formation of insoluble aggregates of the recombinant protein, soluble and functional M. tuberculosis shikimate dehydrogenase could be obtained by repeated cycles of freezing and thawing. Enzyme activity measurements demonstrated that there was approximately a 5-fold increase in specific activity for M. tuberculosis shikimate dehydrogenase. Moreover, the enzyme activity was linearly dependent upon the amount of recombinant protein added to the assay mixture, thus, confirming cloning and expression of functional mycobacterial shikimate.
Avidin-biotin technology is used routinely to purify biotin-containing carboxylases and also proteins that have been chemically coupled to biotin. The 1.3 S subunit of transcarboxylase (TC) studied here is the biotin-containing subunit of TC which not only acts as a carboxyl carrier between the CoA ester sites on the central 12 S subunit of TC and keto acid sites on the outer 5 S subunit of TC but also links the 12 S and 5 S subunits together to form a 26 S multisubunit TC complex. The 1.3 S subunit has been cloned, sequenced, and expressed in Escherichia coli. A method for purifying recombinant 1.3 S subunits from E. coli using avidin (monomeric)-agarose column chromatography has been developed. This affinity-purified 1.3 S was found to be homogeneous by SDS-PAGE, amino acid composition, and N-terminal sequence analysis but had a biotin content of only 28% based on moles of biotin per mole of 1.3 S. This lack of stoichiometry was found to be due to copurification of apo-1.3 S as evidenced by the holocarboxylase synthetase reaction. A procedure for separating the apo- and biotinylated 1.3 S forms using hydrophobic interaction chromatography on an Ether 5 PW column is described. The method is based on the difference in hydrophobicity between apo and biotinylated 1.3 S forms. The copurification of apo and biotinylated forms of 1.3 S on the avidin (monomeric)-agarose column was found to be due to specific interaction with avidin rather than to interaction between apo- and biotinylated 1.3 S forms as demonstrated by the fluorescence quenching studies. The results suggest that the avidin-biotin system by itself may not be sufficient to obtain homogeneous biotinyl proteins as nonbiotinyl protein can also bind avidly to such columns.
Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.
The protein alpha-synuclein plays an important role in many neurodegenerative disorders, referred to as alpha-synucleinopathies, that include, among others, Parkinson's and Alzheimer's diseases. The central region of the wild type protein, known as the non-Abeta component of amyloid plaques (NAC, amino acids 61-95), seems to be responsible for its aggregation process. To structurally characterize this fragment by nuclear magnetic resonance, we produced it by DNA recombinant technology. This technique, unlike chemical synthesis, allows the production of labeled samples (13C, 15N) required for NMR studies. Because the NAC region is very sparingly soluble in aqueous buffer, we cloned a slightly larger portion of alpha-synuclein, alphasyn57-102, with the presence of several charged residues in both extremities of the NAC region. The conformational preferences of purified alphasyn57-102, in solution and bound to SDS micelles, was studied. Our results indicate that the protein is largely unfolded in solution but exhibits a helical conformation in the lipid-associated state. The methodology that we have used in this work for the cloning, expression, and purification of alphasyn57-102 can be easily applied to most small proteins, thus representing a powerful tool for structural NMR analysis of labeled peptides.
Magic roundabout (Robo4) is the fourth recently identified member of the roundabout receptor family. Robo4 is predominantly expressed in embryonic or tumor vascular endothelium and is considered important for vascular development and as a candidate tumor endothelial marker. Much remains unknown about the Robo4 molecule, however, such as its ligands, structure, and the details of its function. Thus, we aimed to establish an expression and purification method for obtaining soluble recombinant human Robo4 (shRobo4) and mouse Robo4 (smRobo4) for use in Robo4 characterization studies. In this work, we expressed the extracellular domain of hRobo4 and mRobo4 in mammalian 293F cells and purified them by two-step chromatography. Based on gel-filtration chromatography and Blue Native polyacrylamide gel electrophoresis, these purified proteins exist as multimers. The shRobo4 and smRobo4 we obtained will be useful in advanced studies to determine the importance of multimerization, identify the ligands, and elucidate the ligand-receptor interactions and Robo4-mediated signaling. The results of these studies will help to elucidate the role of Robo4 in angiogenesis and perhaps eventually contribute to the development of novel vessel-targeting therapies.
The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.
Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.
The xylan binding domain (XBD) and linker sequences (LS) from thermostable and thermophilic Thermomonospora fusca xylanase A (TfxA) was fused to the carboxyl-terminus of a family 11 hybrid xylanase ATx. The constructed chimera (ATxX) was successfully expressed in Pichia pastoris, partially purified to homogeneity, and then characterized in detail. After 96-h 0.25% methanol induction, the xylanase and cellulose activity of ATxX from pPATxX1 transformant culture medium supernatant were 452.1 U/mg and 19.3 U/mg, respectively. SDS-PAGE analysis revealed that the molecular mass of ATxX was about 33.01 kDa. 3.7% ATxX was bound after incubation with 1% microcrystal cellulose at 25°C for 3h, while the ATx did not show cellulose binding-hydrolyzing ability. These results suggested that ATx obtained cellulose binding and hydrolyzing ability by fusing with XBD and LS. Enzymatic studies showed that the temperature and pH optimum of the ATxX xylanase activity were 60°C and pH5.0, respectively, which were the same as that of ATx. The temperature and pH optimum of the ATxX cellulase activity were 60°C and pH6.0, respectively. The major hydrolytic products released by ATxX from birchwood xylan were xylotriose and xylohexaose. Xylooligosaccharides from xylobiose to xylohexaose could be hydrolyzed by ATxX. Mode of action analysis showed that the chimeric ATxX was an endo-acting enzyme. The XBD and LS plays an important role in the binding and hydrolyzing of xylanase to insoluble substrates.
Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.
Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.
To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 degrees C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 degrees C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0-10.0) and much less affected by acid/base conditions. After incubation at pH 3.0-10.0, 25 degrees C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.
A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3-4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11beta-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90-95% pure and exhibited dehydrogenase and reductase activities with K(M) values in agreement with those reported in the literature.
Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).
A novel glucose oxidase (GOX), a flavoenzyme, from Penicillium sp. was isolated, purified and partially characterised. Maximum activities of 1.08U mg(-1)dry weight intracellular and 6.9U ml(-1) extracellular GOX were obtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI's of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148kDa, consisting of two equal subunits with molecular weight of 70k Da. The enzyme displayed a temperature optimum between 25 and 30 degrees C, and an optimum pH range of 6-8 for the oxidation of beta-d-glucose. The enzyme was stable at 25 degrees C for a minimum of 10h, with a half-life of approximately 30 min at 37 degrees C without any prior stabilisation. The lyophilized enzyme was stable at -20 degrees C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5U mg(-1); Km, 18.4mM; kcat, 741 s(-1) and kcat/Km, 40 s(-1)mM(-1). Stability at room temperature, good shelf-life without stabilisation and the neutral range for the pH optimum of this GOX contribute to its usefulness in current GOX-based biosensor applications.
Bovine beta-lactoglobulin (BLG) has been widely used as a model system to study protein folding and aggregation and for biotechnology applications. Native BLG contains two disulfide bonds and one free cysteine at position 121. This free thiol group has been shown to be responsible for the irreversibility of BLG denaturation in vitro, but nothing is known about its relevance during protein folding inside the cell. Here, we report the expression of soluble wild type recombinant BGL in Escherichia coli cells at about 109 mg rBLG/g wet weight cells and a comparison between the aggregation of wt BLG and its variant C121S upon intracellular expression. We show that in E. coli C121SBLG is more prone to aggregation than the wild type protein and that their different behavior depends on the oxidation of disulfide bonds. Our results underline the key contribution of the unpaired cysteine residue during the oxidative folding pathway and indicate BLG as a useful tool for the study of protein aggregation in vivo.
HPSEC on a G2000 SW column of an osmotic shock fluid obtained from an hGH-secreting E. coli strain. A parallel run of the First International Standard of rec-hGH (88/624) indicated a t R of 15.2 min; mAbs, milliabsorbance units with detector wavelength at 220 nm.
A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.
As HuGM-CSF and huIL-6 seem to have synergistic and complementary actions, researchers have proposed that fusion proteins incorporating these two cytokines could show increased biological activity, especially in terms of hematopoietic function. Here, we sought to obtain a functional GM-CSF/IL-6 fusion protein and to investigate its biological activities in vitro. A novel construct encoding a fusion protein of huGM-CSF (9-127) and IL-6 (29-184) was generated in the pBV220 expression vector by step-by-step cloning. Amino acids 1-8 of huGM-CSF and amino acids 1-28 of huIL-6 were deleted by PCR. The mutant huGM-CSF (9-127) and huIL-6 (29-184) cDNAs were linked via a linker sequence encoding 15 amino acid residues (G-G-S-G-S)3. Direct sequencing was used to confirm the validity of the desired construct, and the fusion protein was expressed in Escherichia coli host strain BL21 (DE3) in the form of inclusion bodies (IBs). The expression level was more than 25% of the total cell lysate, and a novel purification and refolding strategy was used to isolate the fusion protein product. Inclusion bodies were purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding by Sephacryl S-200. The renatured fusion proteins were obtained at a purity of >95%, and the strategy of refolding on the gel filtration column was found to be efficient, with a relative refolding rate of 80%. This entire refolding and purification procedure could be performed within one day and may prove applicable to large-scale purification and refolding of recombinant proteins from IBs in E. coli. This new method was used to obtain huGM-CSF (9-127)/IL-6 (29-184) fusion protein with high purity and biological activity. MTT assays in TF-1 and B9 cell lines showed that the specific biological activity of huGM-CSF was 1.14+/-0.10 x 10(8) U/mg, and that for huIL-6 was 1.89+/-0.11 x 10(7) U/mg. The fusion protein exhibited enhanced huGM-CSF, but similar huIL-6 biological activities compared with those of either GM-CSF or IL-6 alone. This suggests that our novel huGM-CSF (9-127)/IL-6 (29-184) fusion protein may hold future promise as a therapeutic agent.
Interleukin-13 is a cytokine which is secreted by activated T lymphocytes and primarily impacts monocytes, macrophages, and B cells. A synthetic gene coding for human interleukin-13 has been prepared and cloned into expression vector pEE12. The construct was transfected into NS-O cells, which showed stable expression of the recombinant protein. A four-step purification procedure consisting of S-Sepharose, Q-Sepharose, hydroxyapatite, and Sephacryl-100 chromatographies yielded bioactive interleukin-13 of > 98% purity. The purified protein was structurally characterized. The extinction coefficient at 280 nm was determined to be 5678 M-1 cm-1. Amino acid sequencing confirmed that the N-terminus of the purified protein was intact. Electrospray mass spectrometric analysis, size-exclusion chromatography, and SDS-PAGE revealed that the biologically active protein is monomeric and unglycosylated. Mass spectrometry and a chemical assay for free sulfhydryls indicated that the four cysteine residues of interleukin-13 are involved in two intramolecular disulfide bonds. The circular dichroism spectrum confirms that interleukin-13 belongs to the alpha-helical family of cytokines. A biologically inactive covalent trimer also forms in the cell culture, but can be separated from the monomer by the hydroxyapatite and size-exclusion chromatographies. These data indicate that human interleukin-13 retains many structural similarities to human interleukin-4, from which it arose by a gene duplication event.
Top-cited authors
David Waugh
  • National Cancer Institute (USA)
Yifeng Li
Tsutomu Arakawa
Nicola A Burgess-Brown
  • University of Oxford
Opher Gileadi
  • University of Oxford