Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

汉滩病毒核蛋白的分段表达及抗原表位分析

在大肠杆菌中对汉滩病毒Ｓ基因４种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的４种ＧＳＴ－ＮＰ融合蛋白均以不溶性包含体形式存在于茵体细胞内,表达量分别占菌体蛋白总量的２９－３６％,分子量分别约为７２ｋＤ、６６ｋＤ、５４ｋＤ和４４ｋＤＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔ显示５４ｋＤ和７２ｋＤ融合蛋白用酶标记汉滩病毒ＮＰＭｃＡｂｌＡ８和抗ＧＳＴ ＭｃＡｂ ３Ｃ１１染色呈阳反应。６６ｋＤ和４４ｋＤ融合蛋     

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.     

Expression of overlapping PreS1 fragment recombinant proteins for the determination of immunogenic domains in HBsAg PreS1 region

The virion of the hepatitis B virus (HBV) is a sphericalparticle of 42-nm diameter whose envelope contains threerelated surface glycoproteins called the large (L), middle(M) and small (S) proteins.All these proteins are expressedfrom one open reading frame using three in-frame startsites [1]. The L protein is the translation product of thewhole open reading frame. The M protein lacks the N-terminal amino acid residue 108–119 of Lprotein (the preS1sequence), and the S protein lacks the N…     

Cloning,expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, and purification of soluble recombinant duck interleukin-2

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin. Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which would be used as an immunoadjuvants for enhancing vaccine efficacy.     

A functional raw starch-binding domain of barley alpha-amylase expressed in Escherichia coli

The mature form of barley seed low-pI -amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281–414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.     

High-yield production of human big endothelin-1 by a combination of chemical modification and proteolysis of a fusion protein in Escherichia coli

A protein modification method has been developed for the production of human big endothelin (ET)-1. Production of a large quantity of big ET-1 by the method described here is expected to facilitate future experiments such as X-ray crystallography and nuclear magnetic resonance studies, aimed at understanding the tertiary structure of big ET-1 and its dynamics. The plasmid pETB-50 used for the synthesis carries the gene for a fusion protein consisting of 34-amino acid (aa) residues of an N-terminal portion of -galactosidase and the 38-aa residues of big ET-1. The fusion protein ETB-50P contains an arginine residue in the big ET-1 portion at its second C-terminal site and three lysine residues including the C-terminal site in the -galactosidase portion, all of which are susceptible to trypsin. Tryptic digestion of the fusion protein quantitatively produced big ET-1 (1–37), which is depleted in the C-terminal serine. However, a treatment of the fusion protein with 1,2-cyclohexanedione prior to tryptic digestion gave full-length big ET-1 with N⁷,-N⁸-(1,2-dihydroxycyclohex-1,2-ylene)-arginine. This modification was reversed to the intact arginine residue when the modified big ET-1 was incubated in 0.5 M TRIS-HCI buffer, pH 8.0. Consequently, a combination of such a reversible protein modification and tryptic digestion gave 1.74 mg of recombinant big ET-1 from 2.01 of culture broth. The procedure described here may be applied to produce other arginine-containing peptides from fusion proteins.     

HepG2细胞膜上乙型肝炎病毒前S1蛋白（preS1）结合蛋白的研究

采用pull down技术研究preS1在HepG2细胞膜上的结合蛋白。以原核表达的GST-preS1融合蛋白为探针蛋白,与生物素标记的HepG2细胞裂解液进行pull down试验分离与preS1结合的膜蛋白。Western blot结果显示HepG2细胞膜上有一大小约110kDa蛋白（p110）与preS1结合。通过对比实验证明该蛋白具有较好的组织特异性和种属特异性。研究结果显示该蛋白是HepG2细胞膜上与preS1结合的蛋白,可能与HBV的早期感染过程有关。     

乙型肝炎病毒表面抗原前S区和谷胱甘肽巯基转移酶融合基因在大肠杆菌中的表达

我们构建了谷胱甘肽巯基转移酶（ＧＳＴ）和完整的或部分缺失的乙型肝炎病毒表面抗原前Ｓ区的融合基因,并在大肠杆菌中进行了表达。融合蛋白的产量随着前Ｓ区长度的增加而迅速降低,而且融合蛋白的前Ｓ区有严重的降解,主要降解位点在ｐｒｅＳ１区的ａ．ａ．７５和ｐｒｅＳ２区的ａ．ａ．１３０和ａ．ａ．１６５左右。利用蛋白降解酶系缺陷型菌株进行的研究表明,这种降解酶存在于多个大肠杆菌株中而且和大肠杆菌中的两个主要的蛋白降解酶系Ｌｏｎ和ｈｔｐＲ无关。具有重要生物学功能的前Ｓ区肽段（ｐｒｅＳ１ａ．ａ．１－６５）因含有阻止分泌的滞留顺序而无法在哺乳动物细胞和酵母中大量表达,但滞留顺序的存在并没有影响含有这一肽段的融合蛋白在大肠杆菌中的表达和产物的纯化。ＧＳＴ融合表达系统产量高,纯化快速简便。用这一方法大量表达并得到的这一肽段不仅是研究乙型肝炎病毒的分子生物学的重要材料,而且可以作为新一代乙型肝炎疫苗的主要组成成分。     

KDP胞外502～764位氨基酸基因合成、表达及功能鉴定

应用基因搭桥法及 Taq酶聚合反应合成了编码人血管内皮生长因子受体 - ( h VEGFR- ,KDR)第 50 2～ 764位 2 62个氨基酸的基因片段 .DNA序列分析表明 ,合成的 786bp的基因片段与文献报道的 KDR相应 c DNA序列完全一致 .将该基因与原核融合蛋白表达载体 p GEX- 3X重组 ,在大肠杆菌 JM1 0 9中表达了 GST- KDR2 62融合蛋白 ,表达量约占菌体总蛋白的 35% .表达产物依次经包涵体分离、变性、复性、亲合层析纯化和 Xa因子酶解 ,获得了 KDR2 62目的蛋白纯品 .GST-KDR2 62融合蛋白和纯化产物经 Western blot分析 ,两者均可被 VEGF1 65特异性识别 ,前者分子量约 56k D,后者分子量约 30 k D;这两种蛋白用 VEGF1 65及其抗体进行的 ELISA分析结果均显示阳性 ,并有剂量依赖关系 ,而用 Xa因子酶解 GST- KDR2 62融合蛋白获得的 GST和空载体诱导产物对照均为阴性 .以上结果表明表达的 KDR2 62蛋白可特异性地与 VEGF结合 .     

Expression and purification of soluble recombinant Human Endostatin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.     

A flexible peptide linker enhances the immunoreactivity of two copies HBsAg preS1 (21-47) fusion protein

PreS1 (21-47) region of HBV large surface protein is hepatocyte receptor binding site and the anti-preS1 (21-47) antibody possesses the virus-neutralizing activity and protective effect. It is important to obtain the peptide with higher immunoreactivity on a large scale for detecting the anti-preS1 (21-47) antibody in the sera from HBV infected patients and future vaccine recipients. The expression vector pGEX SLS, which expressed two copies of the preS1 (21-47) peptide connected by a flexible linker (Gly4Ser3) fused to glutathione S-transferase (GST), was constructed. The fusion protein, named GST-SLS, was highly expressed in E. coli and purified by affinity chromatography. Ninety milligrams purified protein can be obtained from 1l of culture. The data in ELISA analysis showed that the immunoreactivity of GST-SLS was enhanced significantly in comparison with GST-S II, a GST fusion protein with two copies preS1 (21-47) linked directly; GST-S I, another GST fusion protein with one copy preS1 (21-47) and preS1 (21-47) synthesized peptide. In addition, GST-SLS has been tried to use in detecting anti-preS1 (21-47) antibody in the sera from HBV infected patients and a satisfied result was gained. Therefore, GST-SLS may have potential to be developed into a new kit for diagnosis and prognosis of hepatitis B (HB) patients.     

Purification and structural analysis of the hepatitis B virus preS1 expressed from Escherichia coli

The preS1 of hepatitis B virus (HBV) is located at the outermost part of the envelope protein and possesses several functionally important regions such as hepatocyte receptor-binding site and virus-neutralizing epitopes. As the first step to understand the structure-function relationship for the preS1 antigen, we have purified the preS1 and performed its structural characterization by circular dichroism (CD) spectroscopy. The preS1 was purified to near homogeneity from bacterially expressed glutathione S-transferase (GST)-preS1 fusion protein by two-step purification, affinity chromatography on glutathione-agarose column, and cation-exchange chromatography on Mono S column. The CD analysis showed that the purified preS1, which was largely unstructured in aqueous solution, acquired a significant (16%) alpha-helical structure when analyzed in 50% trifluoroethanol or 20 mM SDS. The results suggest that the preS1 assumes a mainly unstructured conformation and may form induced secondary structures upon binding to target proteins or under hydrophobic environment.     

Effects of glycine supplement on protein production and release in recombinant Escherichia coli

Summary The effect of glycine supplement to growth media on protein expression and release in a recombinant strain RR1 of E. coli was investigated. Addition of glycine to the growth media in moderate amount (up to 1%) was observed to enhance significantly the release of periplasmic proteins from the cell to the broth. The extracellular activities of the model enzymes -amylase and -lactamase were increased by a factor of 16.3 and 3.8 respectively in the presence of glycine. These activities corresponded to about 50% of the total production for each protein. Furthermore, with glycine supplement the total enzyme activity of both -amylase, -lactamase as well as -galactosidase were increased by a factor of about 2.5. Cell growth characteristics and low extracellular activity of the cytoplasmic protein -galactosidase are indicative that glycine does not cause significant cell-lysis for a concentration below 0.7%.     

庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880 bp的庚型肝炎病毒cDNA在大肠杆菌BL21(DE3)菌株中得到表达。此cDNA被插入到表达质粒pGEX-5X-1中,位于编码日本血吸虫谷胱甘肽硫转移酶（GST）的DNA序列下游,并与GST处于同一阅读框。用乳糖在37℃下诱导表达出以包涵体形式存在的GST-NS53融合蛋白,并用脲溶法提取了该蛋白;在20℃诱导时,表达出的蛋白大部分可溶,用谷胱甘肽Sepharose-4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明,此融合蛋白能被庚型肝炎病人的血清和自制的抗GST血清特异性地识别。用PCgene软件对NS53氨基酸序列的亲水性和抗原决定簇进行了分析。本研究为庚型肝炎ELISA诊断试剂研制打下了基础。     

Biochemical properties of rice adenylate kinase and subcellular location in plant cells

Previously, we characterized nucleotide sequences of two cDNAs encoding adenylate kinase from rice plants (Oryza sativa L.). Each cDNA (Adk-a or Adk-b) was cloned into the expression vector pET 11d-GST to produce GST-AK fusion proteins in Escherichia coli. Recombinant proteins were cleaved by thrombin, and GST-free adenylate kinase proteins were obtained. Enzyme activity profiles of different pH and inhibition effects to the enzyme by Ap5A (adenosine-5-pentaphospho-5-adenosine) indicates that both adenylate kinase proteins have similar biochemical characteristics. Among the nucleoside monophosphates (AMP, CMP, GMP and UMP) investigated, only AMP reacted with ATP. Furthermore, using the antiserum against the rice adenylate kinase proteins, the cellular location of adenylate kinase proteins was examined by immunomicroscopic analysis in combination with a subcellular fractionation method. The results indicated that adenylate kinase proteins were distributed largely in cytosol of rice cells.Abbreviations AK adenylate kinase - IPTG isopropylthio--D-galactoside - Ap5A adenosine-5-pentaphospho-5-adenosine - PEP phosphoenol pyruvate - GST glutathione S-transferase - BSA bovine serum albumin - FITC fluorescein isothiocyanate     

Hyperproduction of a recombinant fusion protein of Staphylococcus aureus V8 protease in Escherichia coli and its processing by OmpT protease to release an active V8 protease derivative

The expression of a recombinant fusion protein including Staphylococcus aureus V8 protease was studied by using Escherichia coli as the host strain. When the mature V8 protease was expressed as a fusion protein with a truncated E. coli \-galactosidase (\-gal97S4D), we could not obtain a sufficient amount of the enzyme because of the toxicity resulting from the expressed protease activity. Synthesis of V8 protease was increased by constructing a sandwich-type fusion protein consisting of \-gal197S4D, a V8 protease derivative with the 56 C-terminal amino acids deleted (V856) and a truncated aminoglycoside-3'-phosphotransferase. This fusion protein was successfully produced as inactive inclusion bodies. To release the V856 protease from the fusion protein, we developed a novel processing method using an endogeneous E. coli OmpT protease, which can recognize the dibasic amino acid residues located in the linker peptides of the fusion protein. After solubilizing the inclusion bodies with urea, the V856 protein was automatically released from the fusion protein by the OmpT protease, which was coprecipitated with the inclusion bodies. The V856 protease thus obtained showed the same enzymatic activity as that of the native V8 protease. We demonstrate in this study that the N-terminal prepro sequence and the C-terminal repeated sequence of this enzyme are not necessary for its enzymatic activity and protein folding.     

A novel interaction partner for the C-terminus of Arabidopsis thaliana plasma membrane H+-ATPase (AHA1 isoform): site and mechanism of action on H+-ATPase activity differ from those of 14-3-3 proteins

Using the two-hybrid technique we identified a novel protein whose N-terminal 88 amino acids (aa) interact with the C-terminal regulatory domain of the plasma membrane (PM) H+-ATPase from Arabidopsis thaliana (aa 847-949 of isoform AHA1). The corresponding gene has been named Ppi1 for Proton pump interactor 1. The encoded protein is 612 aa long and rich in charged and polar residues, except for the extreme C-terminus, where it presents a hydrophobic stretch of 24 aa. Several genes in the A. thaliana genome and many ESTs from different plant species share significant similarity (50-70% at the aa level over stretches of 200-600 aa) to Ppi1. The PPI1 N-terminus, expressed in bacteria as a fusion protein with either GST or a His-tag, binds the PM H+-ATPase in overlay experiments. The same fusion proteins and the entire coding region fused to GST stimulate H+-ATPase activity. The effect of the His-tagged peptide is synergistic with that of fusicoccin (FC) and of tryptic removal of a C-terminal 10 kDa fragment. The His-tagged peptide binds also the trypsinised H+-ATPase. Altogether these results indicate that PPI1 N-terminus is able to modulate the PM H+-ATPase activity by binding to a site different from the 14-3-3 binding site and is located upstream of the trypsin cleavage site.     

Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV

The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)₆], or a combination of GST and (His)₆ [GST-(His)₆] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)₆-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)₆-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)₆-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.