Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli

Expression of fusion protein trypsin-streptavidin (TRYPSA)⁴ in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio--D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation.     

Purification of an L-asparaginase-atrial natriuretic peptide fusion protein expressed in Escherichia coli

A novel fusion protein designed to facilitate protein purification was expressed in Escherichia coli and purified separately by two different chromatography methods. L-Asparaginase from Erwinia chrysanthemi is fused to the N-terminus of a model peptide, alpha-human atrial natriuretic peptide (alpha-hANP). L-Asparaginase was chosen because of its selective affinity for L-asparagine and because of its unusually high isoelectric point(8.6). A gene construction without the L-asparaginase native signal sequence caused expression at a level of 8% of total cell protein, while gene construction with the native signal sequence resulted in over five time less expression. The hybrid protein expressed without the signal sequence was purified from clarified cell lysate byeither L-asparagine affinity chromatography or cation exchange chromatography. After digestion of the fusion protein with factor Xa protease, a peptide with a molecular weight corresponding to the theoretical molecular weight of alpha-hANP was observed by coupled HPLC/mass spectrometry. (c) 1995 John Wiley & Sons Inc.     

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.     

Purification and some properties of glutathione S-transferase from Escherichia coli B.

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.     

Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli.

A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins. The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein. Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation. This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.     

Chlorothalonil-biotransformation by glutathione S-transferase of Escherichia coli

It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al, 2003). GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification. In an attempt to elucidate the relation between chlorothalonil-detoxification and GST, the GST of Escherichia coli was expressed and purified. The drug-hypersensitive E. coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil. The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione. Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST.     

Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5)

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b5 as a GST-cyt b5 fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein.     

Production and purification of Japanese quail ovalbumin as fusion protein with glutathione S-transferase inEscherichia coli

A plasmid encoding a fusion protein interlinked by thrombin recognition sequence between glutathione S-transferase and Japanese quail ovalbumin (without 40 amino acid residues from the 5′-end of the ORF) has been constructed, employing the expression system pGEX-2T. The deglycosylated fusion protein (64 kDa) was purified by affinity chromatography on glutathione agarose beads, analyzed by SDS-polyacrylamide gel electrophoresis, immunochemically detected with antiserum raised against Japanese quail ovalbumin and tested for its stability.     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Expression of selenocysteine-containing glutathione S-transferase in Escherichia coli

Evolution of a probable 'glutathione-binding ancestor' resulting in a common thioredoxin-fold for glutathione S-transferases and glutathione peroxidases may possibly suggest that a glutathione S-transferase could be engineered into a selenium-containing glutathione S-transferase (seleno-GST), having glutathione peroxidase (GPX) activity. Here, we addressed this question by production of such protein. In order to obtain a recombinant seleno-GST produced in Escherichia coli, we introduced a variant bacterial-type selenocysteine insertion sequence (SECIS) element which afforded substitution with selenocysteine for the catalytic Tyr residue in the active site of GST from Schistosoma japonica. Utilizing coexpression with the bacterial selA, selB, and selC genes (encoding selenocysteine synthase, SelB, and tRNA(Sec), respectively) the yield of recombinant seleno-GST was about 2.9 mg/L bacterial culture, concomitant with formation of approximately 85% truncation product as a result of termination of translation at the selenocysteine-encoding UGA codon. The mutations inferred as a result of the introduction of a SECIS element did not affect the glutathione-binding capacity (Km = 53 microM for glutathione as compared to 63 microM for the wild-type enzyme) nor the GST activity (kcat = 14.3 s(-1) vs. 16.6 s(-1)), provided that the catalytic Tyr residue was intact. When this residue was changed to selenocysteine, however, the resulting seleno-GST lost the GST activity. It also failed to display any novel GPX activity towards three standard peroxide substrates (hydrogen peroxide, butyl hydroperoxide or cumene hydroperoxide). These results show that recombinant selenoproteins with internal selenocysteine residues may be heterologously produced in E. coli at sufficient amounts for purification. We also conclude that introduction of a selenocysteine residue into the catalytic site of a glutathione S-transferase is not sufficient to induce GPX activity in spite of a maintained glutathione-binding capacity.     

Continuous processing of fusion protein expressed as an Escherichia coli inclusion body

In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.     

Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli

Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells.     

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.     

Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli

The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.     

蛇毒锯鳞蝰素融合蛋白的发酵与纯化

研究大肠杆菌表达重组蛇毒锯鳞蝰素(Echistatin,Ecs)融合蛋白的发酵和纯化工艺。将Ecs基因插入表达载体pTXB1,转化E.coliBL21(DE3)构建工程菌。对工程菌进行补料分批培养并诱导表达,研究培养基、培养和诱导时间对工程菌生长和目的蛋白表达的影响,几丁质亲和层析纯化Ecs融合蛋白,经DTT裂解后,检测Ecs活性。发酵后菌体湿重可达75g/L,融合蛋白表达量约占总蛋白的35%,重组质粒在BL21宿主菌中传代稳定。亲和层析纯化后,得到Ecs单体,得率为28mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。优化了Ecs融合基因工程菌的发酵和纯化条件,为规模化生产奠定基础。     

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.     

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.     

Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli

The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition.