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Recombinant production, purification and characterization of vessel dilator in E. coli
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Background: Mean inactivation dose is a useful radiobiological parameter for the comparison of human cell survival curves..
Objectives: Given the importance and accuracy of these parameters, in the present study, the radio sensitivity enhancement of colon cancer (HT-29) cells in the presence of gold nanoparticles (GNPs) were studied using the mean inactivation dose (MID)..
Materials and Methods: Naked-GNPs with 50 nm diameters were incubated with HT-29 cells. The cytotoxicity and uptake of these particles on HT-29 cells were assessed. After determining the optimum GNPs concentration, the cells were incubated with gold nanoparticle for 24 hours. The change in the MID value as well as the radio sensitization enhancement under irradiation with 9 MV X-ray beams in the presence of GNPs were evaluated by multiple (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)MTS assay..
Results: Cell survival in the presence of GNPs was more than 90% and the maximum uptake of GNPs was observed at 60 µM of gold nanoparticles. In contrast, in the presence of GNPs combined with radiation, cell survival and MID value significantly decreased, so that the radio sensitization enhancement was 1.4..
Conclusions: Due to the significant reduction in the mean inactivation dose of colon cancer cells in the presence of gold nanoparticles, it seems that GNPs are suitable options to achieve a new approach in order to improve radiotherapy efficiency without increasing the prescribed radiation dose.
Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.
The ribosome’s interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long,
structured 5′ untranslated regions (5′ UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine
how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome’s interactions
with structured standby sites, upstream of Shine–Dalgarno sequences, and show that these interactions can modulate translation
initiation rates by over 100-fold. We find that an mRNA’s translation initiation rate is controlled by the amount of single-stranded
surface area, the partial unfolding of RNA structures to minimize the ribosome’s binding free energy penalty, the absence
of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first
principles and a four-parameter free energy model to accurately predict the ribosome’s translation initiation rates for 136
synthetic 5′ UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments
confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical
mechanism for observed context effects and long-range post-transcriptional regulation.
The expression of genes, both coding and noncoding, can be significantly influenced by RNA structural features of their corresponding transcripts. There is by now mounting experimental and some theoretical evidence that structure formation in vivo starts during transcription and that this cotranscriptional folding determines the functional RNA structural features that are being formed. Several decades of research in bioinformatics have resulted in a wide range of computational methods for predicting RNA secondary structures. Almost all state-of-the-art methods in terms of prediction accuracy, however, completely ignore the process of structure formation and focus exclusively on the final RNA structure. This review hopes to bridge this gap. We summarize the existing evidence for cotranscriptional folding and then review the different, currently used strategies for RNA secondary-structure prediction. Finally, we propose a range of ideas on how state-of-the-art methods could be potentially improved by explicitly capturing the process of cotranscriptional structure formation.
A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.
The Ribosome Binding Site (RBS) Calculator is a design method for predicting and controlling translation initiation and protein expression in bacteria. The method can predict the rate of translation initiation for every start codon in an mRNA transcript. The method may also optimize a synthetic RBS sequence to achieve a targeted translation initiation rate. Using the RBS Calculator, a protein coding sequence's translation rate may be rationally controlled across a 100,000+ fold range. We begin by providing an overview of the potential biotechnology applications of the RBS Calculator, including the optimization of synthetic metabolic pathways and genetic circuits. We then detail the definitions, methodologies, and algorithms behind the RBS Calculator's thermodynamic model and optimization method. Finally, we outline a protocol for precisely measuring steady-state fluorescent protein expression levels. These methods and protocols provide a clear explanation of the RBS Calculator and its uses.
Motivation: RBSDesigner predicts the translation efficiency of existing mRNA sequences and designs synthetic ribosome binding sites (RBSs)
for a given coding sequence (CDS) to yield a desired level of protein expression. The program implements the mathematical
model for translation initiation described in Na et al. (Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences
with a desired expression level in prokaryotes. BMC Syst. Biol., 4, 71). The program additionally incorporates the effect on translation efficiency of the spacer length between a Shine–Dalgarno
(SD) sequence and an AUG codon, which is crucial for the incorporation of fMet-tRNA into the ribosome. RBSDesigner provides
a graphical user interface (GUI) for the convenient design of synthetic RBSs.
Availability: RBSDesigner is written in Python and Microsoft Visual Basic 6.0 and is publicly available as precompiled stand-alone software
on the web (http://rbs.kaist.ac.kr).
Contact: dhlee{at}kaist.ac.kr
Within the emerging field of synthetic biology, engineering paradigms have recently been used to design biological systems with novel functionalities. One of the essential challenges hampering the construction of such systems is the need to precisely optimize protein expression levels for robust operation. However, it is difficult to design mRNA sequences for expression at targeted protein levels, since even a few nucleotide modifications around the start codon may alter translational efficiency and dramatically (up to 250-fold) change protein expression. Previous studies have used ad hoc approaches (e.g., random mutagenesis) to obtain the desired translational efficiencies for mRNA sequences. Hence, the development of a mathematical methodology capable of estimating translational efficiency would greatly facilitate the future design of mRNA sequences aimed at yielding desired protein expression levels.
We herein propose a mathematical model that focuses on translation initiation, which is the rate-limiting step in translation. The model uses mRNA-folding dynamics and ribosome-binding dynamics to estimate translational efficiencies solely from mRNA sequence information. We confirmed the feasibility of our model using previously reported expression data on the MS2 coat protein. For further confirmation, we used our model to design 22 luxR mRNA sequences predicted to have diverse translation efficiencies ranging from 10-5 to 1. The expression levels of these sequences were measured in Escherichia coli and found to be highly correlated (R2 = 0.87) with their estimated translational efficiencies. Moreover, we used our computational method to successfully transform a low-expressing DsRed2 mRNA sequence into a high-expressing mRNA sequence by maximizing its translational efficiency through the modification of only eight nucleotides upstream of the start codon.
We herein describe a mathematical model that uses mRNA sequence information to estimate translational efficiency. This model could be used to design best-fit mRNA sequences having a desired protein expression level, thereby facilitating protein over-production in biotechnology or the protein expression-level optimization necessary for the construction of robust networks in synthetic biology.
During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that translates
the newly synthesized transcript. Our in vivo measurements show that the overall elongation rate of transcription is tightly
controlled by the rate of translation. Acceleration and deceleration of a ribosome result in corresponding changes in the
speed of RNAP. Moreover, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome
progression, and the rate of transcription. The stimulating effect of a ribosome on RNAP is achieved by preventing its spontaneous
backtracking, which enhances the pace and also facilitates readthrough of roadblocks in vivo. Such a cooperative mechanism
ensures that the transcriptional yield is always adjusted to translational needs at different genes and under various growth
conditions.
Microbial engineering often requires fine control over protein expression--for example, to connect genetic circuits or control flux through a metabolic pathway. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Experimental validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.
Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.
An expression vector (pSJyub-5) was constructed which contained five repeats of the "yeast ubiquitin gene" regulated by a heat-inducible lambda PL promoter. The vector, when expressed in Escherichia coli, produced a penta-ubiquitin of approximately 42 kDa. Purified penta-ubiquitin was found to be as active as the human mono-ubiquitin in the in vitro ATP/ubiquitin-dependent proteolytic assay of the reticulocyte lysate, indicating that the expressed gene product was recognized by the enzymes involved in the ATP/ubiquitin-dependent proteolytic pathway. The inability of penta-ubiquitin to act as a substrate in the pyrophosphate exchange reaction with the ubiquitin-activating enzyme E1 suggested that it had a carboxyl-terminal Asn, in agreement with the nucleotide sequence. In E. coli, the expressed penta-ubiquitin was processed correctly to mono-ubiquitin. The fidelity of processing in E. coli was confirmed by the following observations. The amino acid compositions of the processed mono-ubiquitin and human ubiquitin were similar. The 1H NMR spectrum of peaks representing amide hydrogens of the carboxyl-terminal Arg-74, Gly-75, and Gly-76 of the processed mono-ubiquitin was identical with that of human ubiquitin. The immunoreactivity of processed mono-ubiquitin and human ubiquitin against polyclonal antibodies that recognized epitope(s) only on the carboxyl terminus of ubiquitin were similar. The human and processed mono-ubiquitin were equally active in degrading 125I-bovine serum albumin in the ATP/ubiquitin-dependent in vitro proteolytic assay with reticulocyte lysates. In the pyrophosphate exchange assay with isolated ubiquitin activating enzyme E1, they were also equally reactive, confirming that the expressed and processed ubiquitin contained an intact carboxyl-terminal Gly-76. Purified penta-ubiquitin should prove to be a useful substrate for identifying and isolating processing enzyme(s) involved in the ATP/ubiquitin-dependent proteolytic pathway.
A databank for chemically synthesized genes has been compiled.
A method is described that allows the expression of a stable human proinsulin product in Escherichia coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of beta-galactosidase. In the unfused system, the proinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacterial Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, when multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by cyanogen bromide treatment.
Three native E. coli proteins—NusA, GrpE, and bacterioferritin (BFR)—were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37°C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37°C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-γ were also expressed in E. coli at 37°C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 382–388, 1999.
Atrial natriuretic peptides consist of a family of peptide hormones that are synthesized by three separate genes and then stored as three different prohormones (i. e., 126-amino acid [a. a.]) atrial natriuretic peptide (ANP), 108-a.a. brain natriuretic peptide (BNP), and 126-a.a. C-natriuretic peptide (CNP) prohormones. The gene encoding for the synthesis of the atrial natriuretic peptide prohormone (proANP) consists of three exons and two introns. Exon 1 encodes the signal peptide and the first 16 a. a. of the ANP prohormone. These 16 a. a. form the N-terminus of a peptide hormone named long-acting natriuretic hormone (LANH). A valine-to-methionine substitution in LANH results in a 2-fold increased incidence of strokes in humans. Exon 2 of the proANP gene encodes for three peptide hormones, i. e., vessel dilator, kaliuretic hormone, and ANP. Each of the proANP gene products have vasodilatory, diuretic, natriuretic, and/or kaliuretic properties. Stretch, glucocorticoids, thyroid hormone(s), mineralocorticoids, and calcium enhance proANP gene expression. Enhanced proANP gene expression is found in congestive heart failure, hypertension, and cirrhosis with ascites. The proANP gene is present with invertebrates and plants as well as in humans and other vertebrates.
Studying on fluid induced vibration of a centrifugal pump through volute path and impeller path is valuable and instructive for vibration reduction. Here, the transient response of a motor-pump-workbench FE medal was analyzed with the excitation of fluid force on the interior surface of the centrifugal pump calculated using CFD. The response of a impeller-shaft- support- workbench rotordynamic model was also analyzed with Newmark-β algorithm under the action of gross fluid force and torque on the impeller calculated using CFD. At last, the vibrations incited through the two paths were compared. The results showed that the fluid presssure pulsation on the interior surface of the volute is a wide frequancy-band vibration exitation source, and the vibration of each mode of the centrigugal pump system is excited by the fluid pressure pulsation; the maximum peak of the amplitude spectrum of the vibration displacement of the centrifugal pump workbench incited by fluid force through impeller path appears at the impeller-rotation frequency, while the spectrum maximum peak of the vibration acceleration of the workbench appears not at the blade passing frequency but at the flow channel passing frequency; the vibration of the workbench incited by fluid force through volute path is far less than that through impeller path, so the impeller-shaft-support-workbench path is the main way for fluid induced centrifugal pump workbench vibration.
Glucagon like peptide-1 plays an important role in the regulation of postprandial insulin release. Here we used the split DnaB mini-intein system to produce recombinant human GLP-1/7–36 (rhGLP-1) in Escherichia coli. The C-terminal domain of DnaB mini-intein (IntC) was genetically fused at the N-terminus of rhGLP-1 to produce IntC-GLP-1. IntC-GLP-1 and N-terminal domain of DnaB mini-intein (IntN) protein were prepared in denatured buffer of pH 8.0. IntC-GLP-1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC-GLP-1 at the molar ratio of 1:2. Then rhGLP-1 was released from IntC-GLP-1 via inducible C terminal peptide-bond cleavage by shifting pH from pH 8.0 to 6.6 at 25°C for 24-h incubation. Then the supernatant was applied to a Ni-Sepharose column and the pass through fraction was collected. About 5.34 mg of rhGLP-1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3300.45, which was equal to the theoretical value of GLP-1/7–36. The glucose-lowering activity of rhGLP-1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP-1 without using enzyme or chemical reagents, which could also be used for other similar peptides.This article is protected by copyright. All rights reserved
A method for recombinant production of a polypeptide, exemplified by the therapeutic peptide MBP8298, by recombining and optimizing the nucleic acid encoding said polypeptide, expressing said nucleic acid in a microbial host cell, isolating said polypeptide from the host, and releasing embedded polypeptide from fusion partners or peptide concatamers are explained. Such method may provide increased production or simplified downstream processing for the polypeptide of interest.
Summary Ovarian cancer has the highest mortality rate among all gynecological malignancies with an estimated 20,180 new cases and 15,310 deaths in the United States in 2006. Thus, for every four women diagnosed with ovarian cancer there were three deaths from ovarian cancer in 2006 in the United States. Four hormones synthesized by one gene in the heart i.e. atrial natriuretic peptide (ANP), vessel dilator, long acting natriuretic peptide (LANP), and kaliuretic peptide were evaluated with dose response (1 to 100 ∝M) experiments for the ability to decrease the number of human ovarian carcinoma cells in vitro and their DNA synthesis. There was an 81%, 74%, 71%, 70% and 53% increase in cancer cell death of ovarian adenocarcinoma cells within 24 hours secondary to vessel dilator, kaliuretic peptide, ANP, LANP, and their intracellular mediator cyclic GMP, respectively (each at 100 ∝M) (p
For Abstract see ChemInform Abstract in Full Text.
We describe a method to construct tandem repeats of coding sequences for polypeptides interspersed by single methionine residues and terminating in a His(6) tag that can be purified by Ni chelate chromatography and then cleaved by cyanogen bromide (CNBr) into homogeneous peptide units, Annealing and unidirectional ligation of complementary 42mer oligonucleotides encoding the 13 amino acid residue yeast alpha-mating factor (alpha F) followed by ligation of the oligomerized 42mers into a pET vector placed the alpha-factor tandem repeats downstream of the ketosteroid isomerase (KSI) gene and upstream of a His(6) cassette. A KSI-(alpha F)(5)-His(6) fusion was overproduced in Escherichia coli, purified by Ni chelate chromatography, and then cleaved with CNBr to release insoluble KSI, the His(6) tail, and the alpha-factor peptide units, each terminating with homoserine (HS) lactone. HPLC yielded pure peptide in a yield of 56 mg/L. The alpha-factor-HS(lactone) could be ammonolyzed or hydrolyzed to yield alpha-factor-HS-amide or alpha-factor-HS, respectively. The alpha-factor-HS peptide had similar biological potency as authentic alpha-factor in yeast cell arrest assays. The alpha-factor-HS(lactone) was also reacted with a number of other compounds including analogs of fluorescein, dansyl, and biotin to produce alpha-factor peptides derivatized exclusively at the C-terminus. To test the ability of the expression system to produce longer peptides, 60-67 amino acid residue peptides encoding the Gla domain of profactor IX (FIXQS, FIXQS-His(6)) were also produced. Yields of 50-55 mg/L of pure FIXQS-His(6) and FIXQS-HS(lactone) were obtained.
The heart is a sophisticated endocrine gland synthesizing the atrial natriuretic peptide (ANP) prohormone which contains four peptide hormones, namely atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long-acting natriuretic peptide, which decrease up to 97% of human pancreatic, breast, colon, prostate, kidney and ovarian carcinomas, as well as small-cell and squamous cell lung cancer cells within 24 hours in cell culture. In vivo these four cardiac hormones eliminate up to 80% of human pancreatic adenocarcinomas, up to two-thirds of human breast cancers, and up to 86% of human small-cell lung cancers in athymic mice. Their anticancer mechanism(s) target the Rat sarcoma bound guanosine triphosphate (RAS)-mitogen activated protein kinase kinase 1/2 (MEK1/2)-extracellular signal related kinase 1/2 (ERK1/2) kinase cascade in cancer cells. These four cardiac hormones inhibit up to 95% of the basal activity of Ras, 98% of the phosphorylation of MEK1/2 kinases and 96% of the activation of basal activity of ERK1/2 kinases. They also completely block the activity of mitogens such as the ability of epidermal growth factor to stimulate ERK and RAS. In addition to inhibiting these mitogen-activated protein kinases (MAPKs) they also inhibit MAPK9, i.e. c-Jun-N-terminal kinase 2. These multiple kinase inhibitors are cytotoxic and cause cell death of cancer cells but not of normal cells.
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. Ubiquitin is a 76-residue protein whose covalent conjugation to other proteins plays a role in many biological processes, including cell growth and differentiation. I discuss the current understanding of the N-end rule pathway.
Three native E. coli proteins—NusA, GrpE, and bacterioferritin (BFR)—were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37°C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37°C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-γ were also expressed in E. coli at 37°C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 382–388, 1999.
Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and
has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide
to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was
expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification
by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase.
Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain
protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED50) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to
identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient
generation of high yield Nesiritide and enabled ease of purification.
Keywordsbrain natriuretic peptide–nesiritide–expression–bioprocess–purification–characterization
A vector system has been designed for obtaining high yields of polypeptides synthesized in Escherichia coli. Multiple copies of a synthetic gene encoding the neuropeptide substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) have been linked and fused to the lacZ gene. Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide (CNBr). The isolated multimeric SP fusion protein was converted to monomers of SP analog, each containing a carboxyl-terminal homoserine lactone (Hse-lactone) residue (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse-lactone), upon treatment with CNBr in formic acid. The Hse-lactone moiety was subjected to chemical modifications to produce an SP Hse amide. This method permits synthesis of peptide amide analogs and other peptide derivatives by combining recombinant DNA techniques and chemical methods.
A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2t, residues 196–464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2t production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2t promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2t and of MBP–recROP2t led respectively to the complete degradation or to the truncation of the recROP2t moiety. However, recROP2t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.
As the field of synthetic biology is developing, the prospects for de novo design of biosynthetic pathways are becoming more and more realistic. Hence, there is an increasing need for computational tools that can support these efforts. A range of algorithms has been developed that can be used to identify all possible metabolic pathways and their corresponding enzymatic parts. These can then be ranked according to various properties and modelled in an organism-specific context. Finally, design software can aid the biologist in the integration of a selected pathway into smartly regulated transcriptional units. Here, we review key existing tools and offer suggestions for how informatics can help to shape the future of synthetic microbiology.
Unlabelled:
Within 24h four cardiac hormones, i.e., vessel dilator, kaliuretic peptide, atrial natriuretic peptide, and long acting natriuretic peptide decrease the number of human glioblastoma cells 75%, 68%, 67%, and 65% while Dendroaspis (green mamba) peptide caused a 17% decrease when each were utilized at 100 microM. The four cardiac hormones decreased DNA synthesis 65-87% and increased cyclic GMP 1.3- to 3.8-fold in the glioblastoma cells. Natriuretic peptide receptors (NPR)-A and -C were present.
Conclusion:
four cardiac hormones eliminate up to 75% of glioblastoma cells via cyclic GMP-mediated up to 87% decrease in DNA synthesis.
The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.
Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.
Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, long acting natriuretic peptide and kaliuretic peptide, and the kidney hormone urodilatin have anticancer effects in vitro.
These cardiac hormones and urodilatin were infused subcutaneously for 28 days with weekly fresh hormones since they lose biological effects at body temperature for more than a week at 0.3 nm kg(-1) body weight in athymic mice bearing human small-cell lung carcinomas.
Long acting natriuretic peptide, vessel dilator, kaliuretic peptide, atrial natriuretic peptide and urodilatin eliminated 86%, 71%, 57%, 43% (P < 0.001 for the cardiac hormones) and 25% (P < 0.05; urodilatin) of the human small-cell lung carcinomas. The treated small-cell lung carcinomas that were not cured grew rapidly, similar to the untreated controls, whose volume was 7 fold larger in 1 week, 18-fold increased in 2 weeks, 39-fold increased in 3 weeks, 63-fold increased in 1 month and 97-fold increased in volume in 6 weeks. One vessel dilator treated small-cell lung carcinoma animal developed a large tumour (8428 mm3 volume) on treatment and this tumour was eliminated with utilizing atrial natriuretic peptide and then long acting natriuretic peptide sequentially.
Four cardiac hormones eliminate up to 86% of human small-cell lung carcinomas in athymic mice. Urodilatin can also eliminate small-cell lung carcinomas but at a lower cure rate of 25%. Unresponsive lesions can be eliminated by utilizing different hormones synthesized by the atrial natriuretic peptide gene in a sequential manner.
A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene
was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric
plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In
vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This
represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.
The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS)
Although associated primarily with the cardiovascular system, atrial natriuretic factor (ANF) has been found to increase the magnitude of duodenal contractions and may play a role in salt and water absorption across gastrointestinal epithelium. Because secretory diarrhea and increased peristalsis are commonly associated with conditions related to hypergastrinemia, we examined an animal model of hypergastrinemia (fundusectomy) to evaluate a possible role for ANF. Sprague-Dawley rats underwent either fundusectomy or sham operation. Circulating levels of gastrin (1085 +/- 105 vs 59 +/- 5 pg/ml), ANF (209 +/- 50 vs 59 +/- 10 pg/ml), and pro-ANF 1-98 (786 +/- 80 vs 599 +/- 49 pg/ml) were elevated significantly 3 months after fundusectomy versus control animals. The increased levels of ANF and pro-ANF 1-98 correlated with the increased gastrin levels (p less than 0.05). Tissue content of ANF and pro-ANF 1-98 were determined at sequential sites in the stomach and small intestine. In normal rats ANF concentrations were greatest in the small intestine; pro-ANF 1-98 content was similar in all tissues except ileum (increased). In rats that underwent fundusectomy, ANF and pro-ANF 1-98 were markedly increased in duodenum compared with all other tissues. Only duodenum showed a difference in peptide levels between normal rats and rats that underwent fundusectomy, (ANF, 1.5 +/- 0.5 vs 16.7 +/- 2.3 ng/gm; pro-ANF 1-98, 0.6 +/- 0.3 vs 51.2 +/- 36.1 ng/gm). Circulating ANF and pro-ANF 1-98 are increased in rats that have undergone fundusectomy. Our results suggest that duodenum may be the source of these increased levels.
We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon.
Three human tumor cell lines of widely differing radiosensitivity were used to examine the characteristics of the 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetradium bromide (MTT) assay and to select suitable conditions for its use in assessing the response of cells to ionizing radiation. The optimal concentration of MTT and the time of incubation of the cells with MTT were individualized for each cell line. The relationship between absorbance and cell number was not linear over the wide range of cell numbers that were used. A calibration curve of absorbance against cell number for each cell line was therefore used. Using the assay to quantify metabolically viable cells, growth curves of irradiated and unirradiated cells were constructed on days 0-14 after irradiation. Accurate surviving fractions could be calculated only when cells were in exponential growth. Using this modification to its interpretation, the MTT assay was able to provide a reproducible measure of survival, which compared well with clonogenic cell survival measurements. However, the necessity to optimize conditions of the MTT assay for each cell line severely limits its usefulness in determining the radiosensitivity of cells in primary human tumor cultures.
Over the last ten years it has become increasingly apparent that turnover of particular proteins, under specific conditions, can play as central a role as the transcriptional and translational regulatory mechanisms. This review summarizes our current knowledge of this particular role of proteases responsible for the initial cleavages in most of these interesting degradative processes
A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.
The morphology of active structural and putative ribosomal RNA genes was observed by electron microscopy after lysis of fragile Escherichia coli cells. Conclusions drawn are: most of the chromosome is not genetically active at any one instant; translation is completely
coupled with transcription; the 16S and 23S ribosomal RNA cistrons occur in tandem, in regions which are widely spaced on the chromosome.
Nonsense fragments produced by amber and ochre mutants of the z gene of E. coli are rapidly degraded during exponential growth. Wild type beta-galactosidase is stable in the same conditions.
Bacterial operons concerned with the biosynthesis of amino acids are often controlled by a process of attenuation. The translation product of the initial segment of the transcript of each operon is a peptide rich in the amino acid that the particular operon controls. If the amino acid is in short supply translation is stalled at the relevant codons of the transcript long enough for the succeeding segment of the transcript to form secondary structures that allow the transcribing RNA polymerase molecule to proceed through a site that otherwise dictates termination of transcription. This site is the attenuator; the process is attenuation.
A plasmid containing human preproinsulin cDNA inserted into the endonuclease Pst I site of the ampicillinase gene of plasmid pBR322 was modified by excision of large portions of the ampicillinase-coding region to produce a variety of gene fusion combinations, many of which generated proteins detectable with antisera to insulin or human C peptide. In one case a perfect hybrid of the NH2-terminal half of the leader sequence of ampicillinase (residues -23 to -12) with the human preproinsulin prepeptide beginning at residue -13 was formed; the result was the synthesis and secretion of human proinsulin into the periplasmic space. We have characterized this protein immunologically and also by labeling it biosynthetically or by iodination followed by immunoprecipitation and automated amino acid sequence analysis. It contains the A and B chain regions of insulin as well as specific human C peptide immunodeterminants and is convertible to an insulin-like component by tryptic digestion. These results demonstrate that human proinsulin can be produced by bacteria and that this biosynthetic approach should prove feasible for the production of adequate amounts of human proinsulin for a variety of clinical studies and human insulin for therapeutic purposes.