重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

TRAIL蛋白的体外活性稳定性初步研究

大肠杆菌发酵表达的TRAIL蛋白经过两步纯化可得到纯度95%以上的活性蛋白,得率为40%.天然TRAIL蛋白为同源三聚体形式,在三聚体中心隐蔽结合Zn<'2+>,Zn<'2+>可维持TRAIL蛋白天然结构的稳定性.研究发现在纯化后的重组可溶性TRAIL蛋白中添加Zn<'2+>,并且Zn<'2+>与TRAIL单体的摩尔比...     

SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。     

ATF-PAI2CD融合蛋白的生物学功能

为了解尿激酶型纤溶酶激活物 (urokinase typeplaminogenactivator,uPA)的氨基末端片段 (amino terminalfrag ment,ATF)和纤溶酶激活物抑制剂 2 (plasminogenactivatorinhibitortype 2 ,PAI 2 )突变体PAI 2CD融合蛋白在大肠杆菌的表达情况及进一步研究其生物学活性、将ATF PAI2CD融合基因与大肠杆菌表达载体pLY 4重组得到表达质粒pZWE ATF PAI2CD ,以其转化大肠杆菌JF112 5 ,经温度诱导 ,ATF PAI2CD获得较高水平表达 ,融合蛋白质以包涵体形式存在 ,占菌体总蛋白质的 15 %。包涵体经洗涤、8mol L尿素溶解、稀释复性及离子交换色谱一步分离 ,纯度达 90 % ,分子量与理论值相符。每升发酵液得重组融合蛋白质 5 0mg。经牛奶板法检测具有纤溶抑制活性 ,比活性达 12 0 0 0IU mg ;应用放射竞争法证实融合蛋白质能与肿瘤细胞表面的uPA受体特异性结合。双功能融合蛋白质的纤溶抑制活性与野生型PAI 2 (或PAI 2突变体 ,PAI 2CD)基本一致 ,与肿瘤细胞表面的uPA受体的结合能力同pro uPA。     

枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达

纳豆激酶是一种纤维蛋白溶解酶 ,有望开发成为新型的溶栓药物 .从中国豆豉中分离的具有较强纤溶活性的枯草杆菌DC 2中提取总DNA ,根据纳豆激酶 (NK)基因序列设计引物 ,用PCR法扩增NK基因 .序列分析表明 ,NK基因成熟肽编码区含有 82 5bp ,编码 2 75个氨基酸残基 ,与文献报道的序列分别有 93 4 %和 94 5 %同源性 .将NK基因插入载体pGEX 4T1构建表达质粒pGEX NK ,转化大肠杆菌JM10 9后 ,经 1mmol LIPTG诱导 4h ,发现大量NK融合蛋白表达 ,并形成包涵体 .SDS PAGE分析表明 ,NK融合蛋白作为包涵体的分子量为 5 3kD .凝胶自动扫描结果显示 ,NK融合蛋白约占菌体可溶性蛋白的 2 6 % .     

大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的 34kDIbeB蛋白     

霍乱毒素B亚单位与胰岛素原融合基因的克隆、表达及重组蛋白特性分析

构建了霍乱毒素B亚单位 (CholeratoxinBsubunit,CTB)与胰岛素原 (Proinsulin)的融合基因CTB -PROIN ,将该融合基因克隆到大肠杆菌表达载体pET 30a(+)中 ,获得重组质粒pETCPI,并将该质粒转入大肠杆菌菌株BL21(DE3)中 ;重组菌株经IPTG诱导后 ,其表达产物经过 15 %SDS PAGE分析表明该菌株可以表达融合蛋白 ,其分子量约为 21.6kD ,且主要以包涵体形式存在 ,约占全菌蛋白的 25%。含CTB-PROIN重组蛋白的包涵体经过变性和复性后 ,CTB-PROIN可以在体外自组装成五聚体结构。Westernblotting分析结果表明重组CTB PROIN蛋白可分别被霍乱毒素的抗体和胰岛素的抗体识别 ,说明该蛋白具有霍乱毒素和胰岛素的双重抗原性。同时在体外 ,CTB PROIN蛋白可与神经节苷脂GM1(monosialoganglioside)特异性结合 ,表明了该融合蛋白在体外具有生物活性。这些研究结果为利用原核生物表达系统研制廉价、高效的I型糖尿病口服疫苗奠定了基础。     

人乳头瘤病毒18型L1蛋白的包涵体和可溶性表达及纯化

目的:原核表达系统表达人乳头瘤病毒18型(HPV18)L1蛋白,建立包涵体和可溶性表达的L1蛋白的纯化方法。方法:构建重组表达质粒p GEX-4T-1-HPV18 L1,在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽(GST)琼脂糖凝胶4B亲和层析纯化包涵体蛋白;在菌体中加入三磷酸腺苷(ATP)和3.5 mol/L尿素孵育后,GST 4B亲和层析纯化可溶性蛋白,凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果:SDS-PAGE结果表明,HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达,均产生相对分子质量约为86 000的HPV18 L1-GST融合蛋白。Western印迹结果显示,包涵体纯化后获得的融合蛋白降解条带较多;而可溶性蛋白纯化后获得的融合蛋白未降解,凝血酶酶切后得到HPV18 L1蛋白,可与HPV18 L1蛋白单克隆抗体结合。结论:采用原核系统表达了HPV18 L1-GST融合蛋白,分别建立了包涵体和可溶性蛋白的纯化方法,获得HPV18 L1蛋白,为其进一步应用奠定了基础。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

Signaling active CD95 receptor molecules trigger co-translocation of inactive CD95 molecules into lipid rafts

The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to "mark" inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules.     

Expression and purification of soluble murine CD40L monomers and polymers in yeast Pichia pastoris

The anti-murine CD40L monoclonal antibody MR1 has been widely used in immunology research to block the CD40-CD40L interaction for induction of transplantation tolerance and to abrogate autoimmune diseases. The availability of recombinant CD40L with high binding capacity for MR1 would provide a valuable immunologic research tool. In this study, we constructed the single chain murine soluble CD40L monomer, dimer, trimer and successfully expressed them in yeast Pichia pastoris under the control of the alcohol oxidase promoter. The secreted single chain murine soluble CD40L monomers, dimers, and trimers were initially enriched through histidine tag capture by Ni-Sepharose 6 fast flow resin and further purified on a cation exchange resin. Purity reached more than 95% for the monomer and dimer forms and more than 90% for the trimer. Protein yield following purification was 16 mg/L for the monomer and dimer, and 8 mg/L for the trimer. ELISA analysis demonstrated that the CD40L dimers and trimers correctly folded in conformations exposing the MR1 antigenic determinant.     

Expression, purification, and characterization of recombinant HIV gp140. The gp41 ectodomain of HIV or simian immunodeficiency virus is sufficient to maintain the retroviral envelope glycoprotein as a trimer

Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (K(D) = 1-2 nm) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.     

Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.     

人sCD40L转基因小鼠模型的构建

目的研究阻断CD40-CD40L共刺激信号通路对移植皮肤免疫排斥反应的影响。方法通过RT-PCR技术克隆了呈可溶性表达的CD40L分子胞外区(sCD40L),利用K14启动子构建了sCD40L皮肤特异性表达载体,并利用该载体制备了转基因小鼠。结果所克隆的CD40L胞外区片段其大小及序列符合预期;以哺乳动物表达载体PCI为骨架,通过DNA重组,获得了含K14启动子和sCD40L编码区的皮肤特异性表达载体K14-sCD40L;通过显微注射和胚胎移植,PCR筛选检测:49只G0代小鼠有1只小鼠扩增出特异性条带,阴性对照无条带。结论成功建立sCD40L转基因阳性小鼠。     

alphavbeta5 integrin sustains growth of human pre-B cells through an RGD-independent interaction with a basic domain of the CD23 protein

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the alphavbeta5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg(172), Lys(173), and Cys(174) (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind alphavbeta5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the beta subunit of the alphavbeta5 integrin. The interaction between alphavbeta5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to alphavbeta5.     

Three-dimensional structures of soluble CD4-bound states of trimeric simian immunodeficiency virus envelope glycoproteins determined by using cryo-electron tomography

The trimeric envelope glycoprotein (Env) spikes displayed on the surfaces of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) virions are composed of three heterodimers of the viral glycoproteins gp120 and gp41. Although binding of gp120 to cell surface CD4 and a chemokine receptor is known to elicit conformational changes in gp120 and gp41, changes in quaternary structure of the trimer have only recently been elucidated. For the HIV-1 BaL isolate, CD4 attachment results in a striking rearrangement of the trimer from a "closed" to an "open" conformation. The effect of CD4 on SIV trimers, however, has not been described. Using cryo-electron tomography, we have now determined molecular architectures of the soluble CD4 (sCD4)-bound states of SIV Env trimers for three different strains (SIVmneE11S, SIVmac239, and SIV CP-MAC). In marked contrast to HIV-1 BaL, SIVmneE11S and SIVmac239 Env showed only minor conformational changes following sCD4 binding. In SIV CP-MAC, where trimeric Env displays a constitutively "open" conformation similar to that seen for HIV-1 BaL Env in the sCD4-complexed state, we show that there are no significant further changes in conformation upon the binding of either sCD4 or 7D3 antibody. The density maps also show that 7D3 and 17b antibodies target epitopes on gp120 that are on opposites sides of the coreceptor binding site. These results provide new insights into the structural diversity of SIV Env and show that there are strain-dependent variations in the orientation of sCD4 bound to trimeric SIV Env.     

Differential regulation of soluble and membrane CD40L proteins in T cells

CD40 ligand is an important immunoregulatory protein expressed by T cells. This protein exists as two isoforms, a membrane glycoprotein and a truncated soluble form. Here we demonstrate that membrane and soluble CD40L (sCD40L) are differentially regulated depending upon the activation stimulus. In T cell receptor activated cells, both membrane and sCD40L proteins are expressed and CD28 costimulation further increases their expression. The dissection of TCR generated signals into calcium and PKC-dependent pathways demonstrates that calcium is sufficient to induce membrane CD40L yet insufficient for sCD40L. In contrast, sCD40L is preferentially induced by PKC. Moreover, sCD40L production is blocked by Zn(2+)-dependent metalloproteinase inhibitors while membrane CD40L is concurrently increased. This profile suggests the potential involvement of the ADAM-10 protease which was subsequently shown to cleave membrane CD40L to generate sCD40L. Given the role of sCD40L in numerous disease pathologies and its ability to activate proximal and distal immune responses, the regulated cleavage of CD40L may likely contribute to disease mechanisms.     

CD40 ligand binds to alpha5beta1 integrin and triggers cell signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.     

Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48.

CD2 is a plasma membrane glycoprotein present on T lymphocytes that functions as a cell adhesion molecule (CAM). The CD2 counter-receptor in rodents is the structurally-related CAM CD48. Intercellular adhesion involves the formation of multiple CAM complexes between adhering cells and de-adhesion requires disruption of these complexes. To gain an insight into the initiation and termination of intercellular adhesion, the kinetics and affinity of the rat CD2-CD48 interaction was analysed using a BIAcore instrument, which enables the monitoring of protein binding in real time. A soluble chimeric protein, comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4), bound to immobilized soluble CD2 (sCD2) with a KD of 90 microM. The affinity was also determined in the reverse orientation and sCD2 was shown to bind immobilized sCD48-CD4 with a comparable KD of 60 microM. sCD48-CD4 bound to immobilized deglycosylated sCD2 with a KD of 125 microM, indicating that glycosylation of sCD2 has little effect on the affinity of the interaction. The low affinity was the result of an extremely rapid off-rate constant (K(off) > or = 6 s-1), whereas the on-rate constant was unremarkable (K(on) > or = 10(5) M-1s-1). The kinetic analysis revealed that small amounts of multimeric aggregates of sCD48-CD4 formed in concentrated preparations. Our experience suggests that even low concentrations (< 2%) of these aggregates may be a cause of artifactually high affinity values when analysing low-affinity protein interactions. In conclusion, this study provides the first detailed analysis of the kinetics and affinity of monomeric CAM interactions and suggests that binding between CAMs may be weaker than anticipated.