Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.     

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.     

Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli

The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98 %) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.     

Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

Summary A novel approach to the production of a human glucagon in E. coli is described. The 29 amino acids of human glucagon and pentapeptide linker containing enzyme processing site were fused at the amino terminus to a 57 residue N-terminal portion of the human tumor necrosis factor-alpha (hTNF-). The fusion protein was expressed in the E. coli cytoplasm at levels up to 30% of the total cell protein. Precipitation of the fusion protein near its isoelectric point, specific enterokinase cleavage at the linker site and subsequent HPLC purification makes this approach suitable for the production of glucagon as well as other relatively small peptides with therapeutic interests.     

Expression of human erythrocyte NADH-cytochrome b5 reductase as an alpha-thrombin-cleavable fused protein in Escherichia coli

Recombinant fused protein containing human erythrocyte NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2.) was produced in Escherichia coli, which was linked to the NH2 terminus of beta-galactosidase of the vector pUC13 via a recognition sequence of alpha-thrombin. Cleavage of purified fused protein with alpha-thrombin yielded the enzyme whose apparent molecular weight (32,000) was the same as the native enzyme. The amino-acid sequence from Phe-1 to Leu-10 was determined to be identical to that of the authentic enzyme. The purified enzyme showed an identical absorption spectrum and similar catalytic properties to the native enzyme. Establishment of the expression system would make it possible to determine the reaction mechanism of the enzyme.     

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose-response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.     

Synthesis of poly(hydroxyalkanoates) by Escherichia coli expressing mutated and chimeric PHA synthase genes

Pseudomonas resinovorans phaC1 _Pre and phaC2 _Pre genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by PCR and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that -hydroxydecanoate (67–75 mol%) and -hydroxyoctanoate (25–33 mol%) are the major monomers of the PHA produced in cells grown on decanoate. Sequence analysis showed that the gene products of phaC1 _Pre and phaC2 _Pre had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved /-hydrolase fold in the carboxy-terminal portion of the proteins. Switching the /-hydrolase folds of phaC1 _Pre and phaC2 _Pre yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1 _Pre and phaC2 _Pre that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the /-hydrolase folds of phaC1 _Pre and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells.     

Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells

The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.     

Optimization of expression of an annexin V-hirudin chimeric protein in Escherichia coli

A human Annexin V-Hirudin chimeric protein, Annexin V-Hirudin C, was expressed in Escherichia coli. A broad range of parameters such as plasmid stability during propogation and expression, expression capacity stability, the culture media, the growth time before induction and the induction duration were examined and optimized. Recombinant Annexin V-Hirudin C was purified from the cell lysate supernatants by ethanol precipitation, DEAE-cellulose chromatography and Sephadex G-75 chromatography, and the purified protein showed dose-dependent thrombin inhibitory activity. The overall production of purified Annexin V-Hirudin C protein is 10 mg/l/OD600.     

High-level expression of soluble viral structural protein in Escherichia coli

Pharmaceutically relevant virus-like particles (VLPs) can potentially be manufactured cheaply and efficiently through in vitro assembly of viral structural protein in cell-free reactors, but a bottleneck for this processing route is the currently low-level expression of soluble viral protein in efficient cell factories such as Escherichia coli (E. coli). Here, we report expression levels of up to 180 mg L(-1) that are achievable from low-cell-density E. coli cultures using a simple and low cost strategy. We investigated effects of host strain, plasmid, inducer concentration, pre-induction temperature and cell density at induction with design of experiment (DOE). The statistical approach successfully identified significant effects and their interactions, and provided insights into the role of codon-usage effects in expression of viral structural protein. In particular, our results support the notion that full codon optimization may be unnecessary to improve expression of viral genes rich in E. coli rare codons; using a strategically modified host cell could provide a simpler and cheaper alternative.     

Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the β (1–4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc–hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc–hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc–hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc–hly may have important potential application as a future safely administered human drug and food additive.     

Production of human tetraspanin proteins in Escherichia coli

Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.     

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12.

We previously characterized a set of lambda dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and lambda dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, alpha-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers alpha-acetolactate and alpha-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase, B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the lambda dilv phage from ilv and phage lambda promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.     

Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli

Bacterial protein secretion is important in the life cycles of most bacteria, in which it contributes to the formation of pili and flagella and makes available extracellular enzymes to digest polymers for nutritional purposes and toxins to kill host cells in infections of humans, animals and plants. It is generally accepted that nonpathogenic laboratory strains of Escherichia coli, particularly K12 strains, do not secrete proteins into the extracellular medium under routine growth conditions. In this study, we report that commonly used laboratory strains secrete YebF, a small (10.8 kDa in the native form), soluble endogenous protein into the medium, challenging the status quo view that laboratory strains do not secrete proteins to the medium. We further show that 'passenger' proteins linked to the carboxyl end of YebF are efficiently secreted. The function of YebF is unknown, but its use as a carrier for transgenic proteins provides a tool to circumvent toxicity and other contamination issues associated with protein production in E. coli.