Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.     

N-glycosylated proteins and distinct lipooligosaccharide glycoforms of Campylobacter jejuni target the human C-type lectin receptor MGL

An increasing number of bacterial pathogens produce an array of glycoproteins of unknown function. Here we report that Campylobacter jejuni proteins that are modified by the N -linked glycosylation machinery encoded by the pgl locus bind the human Macrophage Galactose-type lectin (MGL). MGL receptor binding was abrogated by EDTA and N -acetylgalactosamine (GalNAc) and was successfully transferred to Escherichia coli by introducing the C. jejuni pgl locus together with a glycan acceptor protein. In addition to glycoproteins, C. jejuni lipooligosaccharide with a terminal GalNAc residue was recognized by MGL. Recombinant E. coli expressing the C. jejuni pgl locus in the absence of a suitable glycan acceptor protein produced altered lipopolysaccharide glycoforms that gained MGL reactivity. Infection assays demonstrated high levels of GalNAc-dependent interaction of the recombinant E. coli with MGL-transfected mammalian cells. In addition, interleukin-6 production by human dendritic cells was enhanced by C. jejuni lacking N -linked glycans compared with wild-type bacteria. Collectively, our results provide evidence that both N -linked glycoproteins and distinct lipooligosaccharide glycoforms of C. jejuni are ligands for the human C-type lectin MGL and that the C. jejuni N -glycosylation machinery can be exploited to target recombinant bacteria to MGL-expressing eukaryotic cells.     

破骨细胞形成抑制因子TNFR结构区在大肠杆菌中表达、抗体制备及活性测定

破骨细胞形成抑制因子(OPG)对骨的重建与再吸收有重要调节作用,其TNFR结构区行使抑制破骨细胞形成与活性的功能。通过PCR将该区基因片段克隆出来,插入表达载体PET-28a质粒多克隆位点,重组质粒转入大肠杆菌BL21中进行表达,表达产物以包涵体形式存在,包涵体经变性复性后,亲合层析获得重组蛋白。纯化的产物作为抗原免疫兔,得到较高特异性的兔源多克隆抗体。利用小鼠降血钙实验检测变性复性后产物的活性,结果表明该重组蛋白有一定的生物活性。     

Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems

The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.     

Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.     

Virus‐like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli

Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self‐assembled into virus‐like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self‐assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme‐linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2–12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host–pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549–557, 2017     

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.     

产肠毒素大肠埃希菌CfaB亚单位的免疫原性分析

目的表达并纯化产肠毒素大肠埃希菌（Enterotoxigenic Escherichia coli,ETEC）CFA／I定居因子的CfaB亚单位,分析其免疫原性。方法将不含信号肽序列的cfaB基因克隆到pQE一30上,构建重组质粒pQE-30-cfaB并转化EcoliM15,表达融合蛋白6xHis—CfaB,将表达的蛋白纯化和复性后免疫BALB／c小鼠,制备抗CfaB的抗血清,用ELISA法检测抗血清效价。结果6×His—CfaB融合蛋白高效表达,相对分子质量为15．5kD,纯化复性后免疫小鼠所得抗血清效价为1：125000。结论成功构建了高效表达6×His-CfaB融合蛋白的重组质粒,表达的融合蛋白免疫小鼠后获得了高效价的抗血清,为进一步研制以双歧杆菌为表达系统的新型ETEC亚单位口服疫苗奠定了基础。     

重组鼠干细胞因子的原核表达、纯化及生物活性初步分析

目的：研制重组鼠干细胞因子（rmSCF）,并检测其体外生物学活性。方法：提取BALB/c孕龄鼠总RNA,通过RT-PCR扩增mSCF的编码基因,将其克隆至原核表达载体pBV220,转化大肠杆菌DH5α,将重组菌株接种至氨苄青霉素抗性的LB培养基中,扩大培养至5L发酵罐中,42℃诱导表达,经包涵体复性、DEAE-SepharoseFF和Phenyle-SepharoseHP层析纯化,制备rmSCF。结果：构建了pBV220-mSCF原核表达载体,在大肠杆菌中表达了rmSCF,纯化后的rmSCF对类原巨核细胞白血病细胞系的UT-7细胞有明显的刺激作用,比活为1.0×106U/mg。结论：获得了纯度大于95%的rmSCF,将进一步展开其在小鼠体内的药效学研究。     

H1N1型流感病毒神经氨酸酶的原核表达、纯化及免疫原性分析

目的：研究H1N1型流感病毒神经氨酸酶（NA）在原核系统中的表达、纯化方法及其免疫原性。方法：构建了大肠杆菌表达载体pET22b-NA,并转化了大肠杆菌BL21（DE3）;通过SP-Sepharose Fast Flow柱对重组NA进行分离纯化,并用Sephadex G-25柱对SP柱后获得的NA进行柱上复性;用不同剂量的重组NA免疫BALB/c小鼠,并检测其诱导产生的抗体滴度。结果：大肠杆菌表达的NA以包涵体形式存在,通过分离及柱上复性,纯化得到重组NA;NA抗原的免疫原性是剂量依赖的,随着剂量的增加,其免疫原性相应增强,3次免疫后,3μg NA诱导小鼠产生的抗体滴度最高,为1∶7000。结论：大肠杆菌表达的NA具有一定的诱导小鼠产生针对天然NA的抗体的能力,为流感病毒基因工程疫苗研究提供了初步线索。     

Development of pilot scale production process and characterization of a recombinant multiepitope malarial vaccine candidate FALVAC-1A expressed in Escherichia coli

Among the four human malarial species, Plasmodium falciparum causes most of the mortality associated with malaria. Several approaches are being pursued to develop a suitable malaria vaccine since it may be the most effective weapon to fight against malaria. A highly immunogenic, synthetic protein consisting of 21 epitopes from pre-erythrocytic and blood stages of P. falciparum (FALVAC-1A) was constructed and expressed in Escherichia coli. This vaccine candidate was highly immunogenic and induced protective antibodies in rabbits when produced through lab-scale processes in milligram quantities. In order to take this vaccine candidate for further clinical trial, we optimized the process for industrial scale production and purification. Here we describe various methods used in pilot scale production and characterization of FALVAC-1A. A fed-batch cultivation process in a bioreactor at 10-L scale was optimized to express the protein in high yields as inclusion bodies in E. coli cells with the recombinant plasmids. Methods to solubilize, capture and purify the target protein from the inclusion bodies were optimized and the resultant protein was >95% pure based on SDS-PAGE and RP-HPLC. This protein was then refolded and nativity was confirmed by Far-UV CD spectroscopy. Final purified protein was characterized to estimate yield, purity, mass and confirmed to be free of host cell proteins, nucleic acids and bacterial endotoxins. This study confirms that industrial scale clinical grade FALVAC-1A can be produced in a cost-effective manner for clinical trials.     

Expression and DNA binding activity of the tomato transcription factor RIN (ripening inhibitor)

Recently, we have found that the accumulation of ripening inhibitor (RIN) protein increased gradually during tomato fruit ripening. Here, the recombinant protein was expressed in Escherichia coli and affinity-purified. The DNA binding activity of renatured RIN protein was tested by electrophoretic mobility shift assay. The results indicated that an optimal expression and purification system was suitable for obtaining active RIN with DNA binding activity.     

江浙蝮蛇毒碱性磷脂酶A2的溶血位点

用聚合酶链式反应 (PCR)对江浙蝮蛇毒碱性磷脂酶A2 基因进行点突变 ,使对应 34位氨基酸残基由Arg分别突变为Glu和Gln ,将其基因克隆至温敏表达载体pBLMVL2 ,并在大肠杆菌RR1中成功诱导表达 ,表达产物以包涵体的形式存在。对包涵体进行变复性后 ,用凝胶过滤的方法纯化。对纯化后的产物进行酶活力测定及溶血活性测定。实验结果表明 ,突变后的表达产物维持原有磷脂酶A2 活性 ,且溶血活性显著降低 ,表明磷脂酶A2 的34位碱性氨基酸残基在溶血过程具有重要作用     

金属螫合亲和层析纯化和复性重组人铜锌超氧化物歧化酶

将重组人铜锌超氧化物歧化酶（rhCu,Zn-SOD）包含体（经纯化后纯度达80％以上）以稀释法或透析法初步复性后,分别再经金属螯合亲和层析（MCAC）纯化。结果透析复性样品和稀释复性样品经MCAC纯化后的rhSOD比活是各自上柱前样品比活的2．2倍和5．3倍,蛋白回收率分别为64％和25％,同时两者活性回收率皆大于130％。表明目标蛋白rhSOD在经过MCAC纯化的同时又获得进一步复性。SDS-PAGE显示rhSOD为19kD的单一条带,纯度大于95％,比活达到5000u／mg左右,同时NBT生物活性染色鉴定显示出很强的超氧化物歧化酶活性。表明MCAC对于复性不完全的rhCu,Zn-SOD而言是一种纯化和使其进一步复性的简便省时且有效的方法。该方法为以包含体形式表达的基因重组蛋白的纯化和复性提供了新思路。     

Expression of tropomyosin from Blattella germanica as a recombinant non-fusion protein in Pichia pastoris and comparison of its IgE reactivity with its native counterpart

Tropomyosins derived from invertebrates are well-known pan allergens. However, the allergenicities of recombinant tropomyosins are variable. Here, we undertook to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins. Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution, and recombinant tropomyosin was expressed in Pichia pastoris. The allergenicities of the native and recombinant tropomyosins were compared by ELISA inhibition analysis. Native German cockroach tropomyosin showed 18% IgE-binding reactivity to German cockroach sensitized sera. Recombinant tropomyosin was produced without fusion protein and its N-terminus was blocked like that of the native counterpart. The IgE-binding reactivity of the recombinant was found to be comparable to that of native tropomyosin over the concentration range 1-1000 ng/ml by ELISA inhibition testing. Recombinant German cockroach tropomyosin expressed in Pichia pastoris showed better allergenicity than that expressed in Escherichia coli. Other factors in addition to the structural differences of native and recombinant proteins may also influence the IgE reactivities of tropomyosins.     

Expression of Human papillomavirus 16 E7ggg oncoprotein on N- and C-terminus of Potato virus X coat protein in bacterial and plant cells

The E7 oncoprotein from Human papillomavirus type 16 (HPV16) is an attractive candidate for anti-cancer therapeutical vaccine development. In this study, we engineered different fusions of mutagenized coding sequence of E7 oncoprotein (E7ggg) with coat protein of Potato virus X (PVX CP) both on 5'- and 3'-terminus of PVX CP and evaluated the influence of the length of linker (no linker, 4, 15aa) connecting PVX CP and E7ggg on their production. At first the expression in Escherichia coli was conducted to assess the characteristics of the recombinant protein prior to be further produced in plants, that is, resultant proteins were used for screening of their immunological reactivity with antibodies against PVX CP and E7. Fusion proteins successfully expressed in bacteria and plants were partially purified and their reactivity and ability to form virus-like particles were evaluated with anti-E7 antibodies.     

Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli

Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems.     

结核分枝杆菌ESXB-ESXA融合蛋白的表达及其作为检测抗原的初步应用

目的:探索一种大量表达结核分枝杆菌ESXB-ESXA融合蛋白的方法,分析其抗原性,评价其在结核抗体检测中的初步应用。方法:采用PCR方法从结核分枝杆菌基因组中分别扩增esxB和esxA基因,用Linker(G4S)3连接2条目的片段,连接pET-32a(+)载体,构建ESXB-ESXA融合蛋白表达载体;重组表达载体转化宿主细胞大肠杆菌BL21(DE3),IPTG诱导表达、纯化,Western印迹分析重组蛋白的抗原性;建立以融合重组蛋白为抗原的ELISA和胶体金检测方法,分析其在结核病抗体检测中的应用。结果:构建了ESXB-ESXA融合蛋白的表达载体,重组ESXB-ESXA在大肠杆菌中得以高效表达,表达量占菌体总蛋白的70%以上;Western印迹分析表明该重组蛋白具有较好的抗原性;ELISA和胶体金检测结果显示,用重组ESXB-ESXA抗原检测临床结核病患者有较好的特异性。结论:重组ESXB-ESXA融合蛋白表达量高并具有较好的抗原性,可作为结核病抗体检测的备选抗原。     

HIV-1p24蛋白在大肠肝菌中的高效可溶性表达、一步纯化及抗原性分析

合成引物扩增HIV 1p2 4基因 ,并将其克隆到 pQE 30质粒中 ,使其在大肠杆菌E .coliM 15中以IPTG诱导高效表达 ,经SDS PAGE分析 ,该表达产物约占菌体总蛋白 2 0 % ,并且以可溶蛋白的形式存在于细菌裂解液上清之中。经镍离子柱亲和层析一步纯化 ,洗脱产物中 p2 4蛋白纯度达95 %。ELISA分析表明 ,该蛋白可与HIV感染者血清发生特异性免疫反应。以此蛋白交联Sepharose 4B ,亲和层析纯化HIV感染者血清中的抗体 ,用所得抗体与HIV确认试剂反应 ,发现该纯化抗体仅与确认试剂中的 p2 4蛋白反应。上述结果表明在大肠杆菌中已经高效表达了可溶性HIV 1p2 4蛋白 ,该蛋白具有良好的抗原性