Synthesis of bioactive seminalplasmin by expression of a newly constructed fusion gene

A synthetic DNA, carrying the coding sequence for seminalplasmin (SAP), the major basic protein of bull semen, was cloned into the C-terminal part of a shortened, mutated fragment of the lacZ gene (lacZ-MF) of vector pLZPWB1. As a result of the mutation, all methionine as well as cysteine residues are replaced by other amino-acid residues. In the fusion gene lacZ-MF-SAP of the resulting construct pSAP4 the two proteins are linked through a methionine residue. Expression of pSAP4 in E. coli W3110 in the presence of the inducer isopropylthiogalactoside (IPTG) led to production of fusion protein with a yield of approximately 50% of the total proteins synthesized. All SAP-immunoreactive fusion protein was found within the insoluble protein fraction and represented 40% of total proteins produced during expression. The fusion protein was subjected to cyanogen bromide cleavage. The overall yield of crude SAP with a purity of 80% was 10 mg/l of culture. The crude SAP was further purified by calmodulin-Sepharose affinity absorption. Characterisation by protein chemical analysis indicated the identity of recombinant SAP with authentic SAP purified from bull semen.     

Gene fragment polymerization gives increased yields of recombinant human proinsulin C-peptide

A multimerization strategy to improve yields upon recombinant production of the 31-aa human proinsulin C-peptide is presented. Gene fragments encoding the C-peptide were assembled using specific head-to-tail multimerization. DNA constructs encoding one, three or seven copies of the C-peptide gene, fused to a serum albumin binding affinity tag, were expressed intracellularly in Escherichia coli. The three fusion proteins were produced at similar levels (approximately 50 mg/l) and were proteolytically stable during production. Enzymatic digestion by trypsin-carboxypeptidase B treatment of the fusion proteins was shown to efficiently release native C-peptide, as determined by mass spectrometry, reverse-phase chromatography and a radioimmunoassay. The quantitative yields of C-peptide obtained from the three different fusion proteins suggest that this multimerization strategy could provide a cost-efficient production scheme for the C-peptide, and that this strategy could be useful also for production of other recombinant peptides.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95% purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells.     

Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide

In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as     

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).     

Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins

In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

Screening of fusion partners for high yield expression and purification of bioactive viscotoxins

Viscotoxins are small cationic proteins found in European mistletoe Viscum album. They are highly toxic towards phytopathogenic fungi and cancer cells. Heterologous expression of viscotoxins would broaden the spectrum of methods to be applied for better understanding of their structure and function and satisfy possible biopharmaceutical needs. Here, we evaluated 13 different proteins as a fusion partners for expression in Escherichia coli cells: His6 tag and His6-tagged versions of GB1, ZZ tag, Z tag, maltose binding protein, NusA, glutathione S-transferase, thioredoxin, green fluorescent protein, as well as periplasmic and cytosolic versions of DsbC and DsbA. The fusion to thioredoxin gave the highest yield of soluble viscotoxin. The His6-tagged fusion protein was captured with Ni(2+) affinity chromatography, subsequently cleaved with tobacco etch virus protease. Selective precipitation by acidification of the cleavage mixture was followed by cation exchange chromatography. This protocol yielded 5.2mg of visctoxin A3 from 1l of culture medium corresponding to a recovery rate of 68%. Mass spectrometry showed a high purity of the sample and the presence of three disulfide bridges in the recombinant viscotoxin. Proper folding of the protein was confirmed by heteronuclear NMR spectra recorded on a uniformly 15N-labeled sample. Recombinant viscotoxins prepared using this protocol are toxic to HeLa cells and preserve the activity differences between isoforms B and A3 found in native proteins.     

Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.     

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.     

重组类胰岛素样生长因子-Ⅰ的纯化与复性

目的 获得高纯度和高活性的胰岛素样生长因子（Insulin-like growth factor, IGF-1）;方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2＋柱亲和层析, IGF-1纯度达90％以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。     

Protein engineering to optimize recombinant protein purification

Genetic approaches have been used to facilitate purification of recombinant proteins, on both a large and a small scale. Based on developments in three different areas: (i) affinity chromatography; (ii) specific cleavage of fusion proteins and (iii) secretion of fusion proteins, a coupled expression/secretion system was designed. It was further improved by protein engineering. Using a synthetic DNA fragment, encoding two IgG-binding domains derived from staphylococcal protein A, gene products were secreted to the culture medium of Escherichia coli and purified with a one-step affinity procedure. The system has been used for large-scale production of biologically active human peptide hormones, to generate peptides for antibody production and to immobilize proteins on solid supports.     

Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris

As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.     

Two-step metal affinity purification of double-tagged (NusA-His6) fusion proteins

The present purification protocol applies to target proteins that are fused to a double tag, such as NusA-His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet.     

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin–agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.     

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.     

Overexpression of soluble human thymosin alpha 1 in Escherichia coli

Synthesized gene of human thymosin alpha 1 (Tα1) was inserted into pET-28a, pET-9c,pThioHis B, pGEX-2T or pBV222 and then inductively expressed in strains of Escherichia coll. Among the five expression systems, the BL21/pET-28a system provides the highest expression level of fusion protein in a soluble form, which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting fusion protein purified through nickel affinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity by SDS-PAGE. These results indicate the potential of this expression system for high-throughput production of recombinant Tα1.     

重组人胰高血糖素样肽-1突变体的分离纯化及活性分析

对基因工程构建的含人胰高血糖素样肽1(hGLP1)突变体的工程菌株进行诱导表达,分离纯化N末端第二位突变的2GlyhGLP1突变体.IPTG诱导4h,收获的菌体经超声破碎后,裂解液用GlutathioneSepharose4B亲和层析纯化GST2GlyhGLP1融合蛋白,经CNBr裂解、SephadexG25柱脱盐、QAESepharoseFF阴离子交换柱层析和RPC18柱脱盐,得到纯度大于98%的重组2GlyhGLP1.Western印迹分析证实,该突变体可被特异性hGLP1抗体所识别.生物学活性分析表明,2GlyhGLP1具有明显的降血糖活性和促胰岛素分泌活性(P<0.001).