重组人G-CSF双体分子的构建表达及生物活性和结构特性分析

研究目的是分子设计并构建较G—CSF。单体分子具有半衰期更长、生物活性更高的新型重组人G—CSF／G—CSF双体分子(简称G-G),并在原核系统进行高效表达。分别构建pET32／G—G和pET32／G原核表达载体,实现G-G双体融合蛋白在大肠杆菌中的高效表达,利用亲和层析等方法进行蛋白纯化;对该融合蛋白的结构特征诸如等电点、柔性、抗原性及亲水性进行模拟分析;采用MTT法对G-G双体融合蛋白的生物学活性进行测定。结果首次构建成功了G-G双体分子融合蛋白高效表达载体,表达量高于40％,一步亲和层析所获融合蛋白的纯度为80％左右。该融合蛋白的结构特征模拟分析的结果显示,G-CSF双体分子的等电点、柔性、抗原性及亲水性均未显改变。活性测定表明,所构建表达的重组人G-G双体分子能有效刺激G—CSF依赖细胞株NFS-60的增殖,但其刺激效应低于G-CSF的单体和标准品。结果表明,所构建表达的G-CSF的单体和G-G双体分子均可在大肠杆菌中获得高效表达,但G-G双体分子的比活性不及G-CSF单体分子,与预期设想的有差别,其原因正在研究之中。     

霍乱毒素B亚单位与谷氨酸脱羧酶抗原表位肽融合蛋白的表达及鉴定

目的:在大肠杆菌中表达霍乱毒素B单位(CTB)与谷氨酸脱羧酶(GAD)抗原表位肽段(531～545)的融合蛋白。方法:通过PCR技术将CTB基因与GAD基因融合在一起,插入pET22b载体,转化大肠杆菌BL21(DE3)后经诱导获得融合基因的表达;对表达产物进行包涵体复性后,纯化得到融合蛋白分子;对该融合蛋白分子进行了Western印迹和GM1-ELISA分析。结果:表达的融合蛋白的相对分子质量约为14000,Western印迹表明该融合蛋白具有霍乱毒素抗原性;GM1-ELISA实验表明该融合蛋白能够特异性地结合神经节苷酯GM1,表明该蛋白具有与CTB相似的五聚体结构。结论:融合蛋白CTB-GAD的成功表达,为后续动物实验提供了充足的抗原。     

小鼠γ干扰素/上皮生长因子受体结合域融合蛋白的构建及其抗肿瘤活性的提高

采用基因工程和蛋白质工程技术,构建了一种新颖的小鼠γ干扰素/上皮生长因子受体结合域融合蛋白,该蛋白保留了完全的抗病毒活性,达到10~8IU/L菌液;利用~(125)I-mEGF进行的受体结合竞争抑制实验,证明融合蛋白所带有的上皮生长因子结合域有与EGF竞争结合受体的功能;体外抗乳腺癌细胞增殖实验证明融合蛋白具有比干扰素母体分子更明显的抗肿瘤细胞增殖效应;在小鼠B16黑色素瘤模型上,融合蛋白和干扰素母体治疗后的2组小鼠,平均瘤体重量分别为1.6和2.6g,具有统计学差异.表明融合蛋白具有比干扰素母体分子更强的体内肿瘤生长抑制作用.     

胸腺素α原融合蛋白基因表达、纯化与生物学活性

胸腺素α原及胸腺九肽在体内免疫方面有重要作用 .根据大肠杆菌遗传密码的偏爱性 ,设计并人工合成了胸腺素α原及胸腺九肽融合蛋白的特异引物 .以胸腺素α原cDNA为模板 ,应用PCR及分子克隆技术构建了该融合蛋白的编码基因 ,将其插入pBV2 2 0质粒 ,转化大肠杆菌 ,通过温度诱导使该融合蛋白高效表达 ,并对表达产物进行了高效特异性纯化 .运用MTT法初步测定了该融合蛋白的活性 .结果表明 ,该融合蛋白对氢化可的松诱导的胸腺细胞损伤有一定的保护作用 ,为今后进一步研究和应用该融合蛋白奠定了基础     

酿酒酵母ATP合酶δ亚基融合蛋白的构建、表达及生物学功能分析

利用分子克隆手段构建了酿酒酵母ATP合酶δ亚基和流感病毒血凝素(haemagglutinin, HA)标签融合蛋白的表达质粒.该融合蛋白在酿酒酵母细胞中能够正常表达,而且能够互补编码δ亚基的ATP16基因缺失株在利用非发酵性碳源方面的表型缺陷,表明该融合蛋白具有野生型δ亚基的功能.同时,构建了在大肠杆菌细胞中表达该δ亚基的ScAtp16p-His6融合蛋白,并用纯化的融合蛋白在家兔中制备了其多抗血清.结果表明此多抗可以很好地与ScAtp16p-His6和HA-ScAtp16p两种融合蛋白特异性结合.这些研究材料的获得为深入研究ATP合酶的解偶联机制和磷酸化调控机理奠定了基础.     

靶向B细胞并特异结合其分泌IL-10的重组融合蛋白的构建、表达和初步鉴定 *

目的:CD19单链抗体可变区(single-chain fragment variable)sc Fv能特异性识别结合B细胞表面的CD19分子,白细胞介素-10(interleukin-10,IL-10)是调节性B细胞(Breg)的主要特征之一。旨在构建CD19sc Fv和IL-10受体1(IL-10R1)胞外段的重组融合蛋白,拟用该融合蛋白捕捉和封闭Breg细胞分泌的IL-10。方法:CD19sc Fv和IL-10R1胞外段克隆到p ET-28a原核质粒上,E.coli BL21(DE3)工程菌表达蛋白通过镍柱富集和纯化蛋白,SDS-PAGE和Western blot对表达蛋白进行鉴定,Pull down实验验证蛋白与CD19分子和IL-10细胞因子的结合。结果:成功表达CD19sc Fv-IL-10R1融合蛋白,该融合蛋白能在体外同时结合CD19和IL-10分子。结论:CD19sc Fv-IL-10R1融合蛋白在体外可以同时结合CD19分子和IL-10分子,具有潜在应用前景可作为特异性抑制Breg的生物小分子。     

分子内二硫键对HERV囊膜蛋白融合核心结构的影响

合胞素(Syncytin)是一类由人俘获的逆转录病毒囊膜蛋白,与胎盘的形态发生中细胞滋养层到合胞滋养层的分化过程十分相关.Syncytin 与人免疫缺陷病毒I型(HIV-1) 囊膜蛋白(Env)在结构上具有相似的特点,二者可能具有相似的膜融合机制.本文通过PCR对融合核心部位七肽重复区HR1和HR2之间linker中自然存在的一对保守的分子内二硫键进行定点突变,表达纯化该突变蛋白,并进行了相应的结构及稳定性探讨,通过与未突变蛋白的性质比较确证该分子内二硫键在蛋白结构的正确形成及稳定性上起着一定的作用.     

SARS冠状病毒NS融合蛋白的重组表达纯化与免疫学性质分析(英文)

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.     

Fc 融合蛋白在药学领域的研究进展

Fc 融合蛋白是指利用基因工程等技术将某种具有生物活性的功能蛋白分子与Fc 片段融合而产生的新型重组蛋白,其不仅保留了功能蛋白分子的生物学活性,还具有一些抗体的性质,如通过结合相关Fc 受体延长半衰期和引发抗体依赖细胞介导的细胞毒性效应等。对Fc融合蛋白及其在药学领域的研究进展进行了综述。     

大鼠脑红蛋白(NGB)的原核表达、抗体制备及其细胞分布

脑红蛋白 (NGB)是新发现的与脑内氧供应密切相关的分子 .为了检测细胞内脑红蛋白的表达、亚细胞分布从而对该分子进行深入的功能研究 ,成功地将大鼠脑红蛋白基因编码区构建于原核表达载体pGEX 4T 2 ,转化大肠杆菌BL2 1(DE3) ,获得融合表达产物 .对含有融合蛋白的包含体进行溶解和复性 ,用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化 ,通过免疫家兔获得了兔源性抗NGB多克隆抗体 .采用Western印迹分析技术 ,用该抗体检测NGB基因的真核表达产物 ,证明该抗体有较好的针对NGB蛋白的专一性 ,可用于对NGB的结构和功能研究 .同时 ,用该抗体进行免疫组化分析发现 ,正常成年大鼠神经系统中有较多的NGB免疫反应阳性细胞分布 ,提示NGB是与神经系统功能密切相关的重要分子     

C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development

Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.     

重组水蛭素相关肽Hi-lys的表达与纯化(英文)

为开发一种新的有临床应用价值的抗血栓药物,根据水蛭素保持抗凝活性的2 0肽片段,设计并构建了水蛭素相关肽(Hi lys)与天冬酰胺酶C端的融合表达系统.为方便目的肽与融合伙伴的分离,增加了富含带电序列的8肽(KRKRKKSR)及酸敏感的天冬氨酰 脯氨酸(Asp Pro)位点,获得了表达质粒pED P8 Hi lys.将其转化E .coliBL 2 1,玉米浆培养基(kanr)培养,乳糖诱导获得融合蛋白(AnsB C P8 Hi lys)的高效表达.通过细菌裂解、包涵体洗涤、尿素溶解、乙醇沉淀、酸水解和DEAE 纤维素5 2柱层析纯化获得目的肽Hi lys ,用凝血酶测定法测得其抗凝活性为5 0ATU mg .     

Thrombin plasticity

Thrombin is the final protease generated in the blood coagulation cascade. It has multiple substrates and cofactors, and serves both pro- and anti-coagulant functions. How thrombin activity is directed throughout the evolution of a clot and the role of conformational change in determining thrombin specificity are issues that lie at the heart of the haemostatic balance. Over the last 20 years there have been a great number of studies supporting the idea that thrombin is an allosteric enzyme that can exist in two conformations differing in activity and specificity. However, recent work has shown that thrombin in its unliganded state is inherently flexible in regions that are important for activity. The effect of flexibility on activity is discussed in this review in context of the zymogen-to-protease conformational transition. Understanding thrombin function in terms of 'plasticity' provides a new conceptual framework for understanding regulation of enzyme activity in general. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Thrombin plasticity

Thrombin is the final protease generated in the blood coagulation cascade. It has multiple substrates and cofactors, and serves both pro- and anti-coagulant functions. How thrombin activity is directed throughout the evolution of a clot and the role of conformational change in determining thrombin specificity are issues that lie at the heart of the haemostatic balance. Over the last 20 years there have been a great number of studies supporting the idea that thrombin is an allosteric enzyme that can exist in two conformations differing in activity and specificity. However, recent work has shown that thrombin in its unliganded state is inherently flexible in regions that are important for activity. The effect of flexibility on activity is discussed in this review in context of the zymogen-to-protease conformational transition. Understanding thrombin function in terms of ‘plasticity’ provides a new conceptual framework for understanding regulation of enzyme activity in general. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.     

Matrilysin: Expression, purification, and characterization

The expression vector pGEX-2T under the control of the IPTG-inducibletac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin,HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.     

Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition.

We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its [3H]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [3H]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.     

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.     

A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen

Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies.