SPAP2基因的克隆、融合表达及分离纯化

本文构建的表达载体pGex-2T-SPAP2CT在大肠杆菌中表达出可融性蛋白, 其分子量为46 000, 经纯化后得到产率为10%、 纯度大于90%的GST-SPAP2CT蛋白.     

脂双层交插结构对人红细胞膜带3蛋白的作用

本文通过将人红细胞膜的阴离子通道——带3蛋白重组于不同的磷脂囊泡来研究脂双层的交插结构对膜内部蛋白构象及功能的影响。用多粘菌素B或三羟甲基氨基甲烷(Tris~+)来诱导DPPG/band3或DPPG/DMPC/band3囊泡形成脂酰链相互交错对插的脂双层(简称交插结构),通过高灵敏的差示量热扫描来检测脂囊泡的结构特征。荧光研究表明交插的脂双层能够增强重组带3蛋白的内源荧光强度并降低蛋白上色氨酸残基的暴露程度,而且随着脂双层交插结构与非交插结构的相互转变,重组带3的构象也发生可逆变化。通过囊泡对~(35)SO_4~(2-)的转运实验表明交插的DPPG脂双层使带3基本丧失转运功能。     

小鼠B7-H3-Fc融合蛋白的表达及其生物学活性的研究

目的:制备B7-H3-Fc融合蛋白,研究其对T细胞的共刺激作用.方法:首先采用PCR技术分别从pMD19-T/小鼠B7-H3和pMD19-T/hIgG1(Fc)重组载体中扩增出小鼠B7-H3胞外段基因和人IgG1重链Fc恒定区基因.通过重叠PER技术将2段基因连接成B7-H3-Fc,经EcoR I和Bgl Ⅱ双酶切后插入真核表达载体plRES2-EGFP构建成pIRES2-EG-FWB7-H3-Fc重组载体.脂质体法转染CHO细胞,经G418加压筛选能稳定分泌表达小鼠B7-H3-Fc融合蛋白的基因转染细胞,并经Western blot鉴定.该转基因细胞无血清培养后,收集细胞上清、超滤浓缩后行经Protein G柱纯化,获得纯品B7-H3-Fc融合蛋白.通过CCK-8以及ELISA方法检测小鼠B7-H3-Fc融合蛋白对T细胞体外增殖及细胞因子分泌的影响.结果:成功地构建了能稳定表达B7-H3-Fc融合蛋白基因的CHO转基因细胞株,该融合蛋白能够剂量依赖性地促进T细胞体外增殖及IL-2和IFN-γ等细胞因子分泌.结论:本研究提示B7-H3作为重要的共刺激分子,在调节T细胞免疫应答中发挥了正性共刺激作用.     

源于大肠杆菌蛋白的表达、液相色谱复性与纯化新进展

对近两年来源于大肠杆菌（Escherichia coli,E．coli）的蛋白表达和用蛋白折叠液相色谱（protein folding liquid chromatography,PFLC）法对所形成的包涵体目标蛋白的复性并同时纯化的新近发展做了简要的介绍和评述．PFLC法用于包涵体蛋白分离、纯化很广,其特点是除了在色谱柱上将目标蛋白与其他组分分开,还同时要在色谱柱上进行包涵体蛋白折叠．可以说,现代生物技术中所用的大多数有价值蛋白产品的制备仍然有赖于不同机理的液相色谱（Lc）法．而用PFLC法对源于E．coli的蛋白的制备方法更具可塑性和容易达到规模化,其生成本可以成倍地降低．该文主要内容包括了E．coli蛋白的表达及样品前处理、PFLC的实用范围、PFLC的优化、PFLC中的新技术、新设备和新方法、PFLC的分子学机理、应用事例及对未来的展望．     

铜离子与SALI3-2蛋白的相互作用

用1/2 MS固体培养基培养拟南芥幼苗, 研究了CuCl₂对转Sali3-2基因植株生长的影响, 发现转基因植株Sali3-2的表达可提高其耐受Cu²⁺胁迫的能力. 进一步克隆Sali3-2基因, 表达并纯化了缺失信号肽的 SALI3-2 蛋白. 利用固定金属离子亲和色谱(IMAC)分析发现, Cu²⁺ 能够与 SALI3-2 蛋白结合. 通过荧光光谱及圆二色光谱(CD)进一步研究了Cu²⁺ 与 SALI3-2 蛋白间的键合机理. 结果表明, Cu²⁺ 可引起SALI3-2 蛋白内源性荧光猝灭, 其猝灭机制为静态猝灭; Cu²⁺ 与SALI3-2 蛋白的结合常数为8.89×10⁶, 结合位点数为1.6. CD分析显示, Cu²⁺ 与SALI3-2 蛋白的结合未使SALI3-2 蛋白二级结构发生明显改变. 由此推测, 在高浓度铜离子胁迫下, SALI3-2蛋白通过结合一定数量的Cu²⁺使蛋白的构象发生改变, 这可能是SALI3-2蛋白的表达使植物耐受Cu²⁺胁迫能力提高的分子机制之一.     

大鼠肝脏金属硫蛋白的分离纯化和金属硫蛋白亚型-Ⅱα结构域片段的制备及其核磁共振鉴定

Wistar雄性大鼠经腹腔注射CdCl_2诱导其肝脏合成金属硫蛋白。取鼠肝,先经匀浆、热变性、乙醇-氯仿萃取、丙酮沉淀等步骤,后由Sephadex G-75、DEAE-52柱层析,得金属硫蛋白的两亚型Ⅰ和Ⅱ。经原子吸收光谱测定每蛋白分子含有5个镉和2个锌。用枯草杆菌酶割法制备并分离了金属硫蛋白亚型Ⅱ的α结构域片段。还用NMR研究了金属硫蛋白的两个亚型及所得的α结构域片段。     

亲和柱色谱原位酶切法纯化重金属镉结合的金属硫蛋白-3

按照人金属硫蛋白-3(hMT-3)的基因序列,选用大肠杆菌偏爱的密码子合成了全长hMT-3基因,并将其插入大肠杆菌融合表达质粒pALEX的多克隆位点中,在谷胱甘肽-硫-转移酶(GST)下游与GST融合表达。通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导在大肠杆菌表达菌株BL21（DE3）LysS中表达了与重金属离子镉结合的融合蛋白GST-Cd2+-hMT-3。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明融合蛋白主要在超声上清液中。分别通过“先纯化、后酶切”和“亲和柱色谱原位酶切”两种方法纯化了Cd2+-hMT-3,比较了两种方法的纯化效率和得率,表明原位酶切法操作简便,较之“先纯化、后酶切”法减少了洗脱、透析、冻干等步骤,从而也减少了样品的损失,提高了样品的纯度和得率。从摇瓶培养菌液中纯化获得了结合有Cd2+的完整的人金属硫蛋白-3,得率为1.8%。氨基酸组成分析结果表明所获得的Cd2+-hMT-3不含芳香族氨基酸和组氨酸,符合金属硫蛋白的特征;直读电感耦合等离子体发射光谱分析其硫镉原子比为21∶(7.5±0.1),与理论值21∶7基本吻合。     

重组人细胞红蛋白的表达纯化及谱学表征

以可溶性和包涵体两种形式表达纯化得到了重组人细胞红蛋白, 并比较了其谱学特征和热稳定性. 可溶性蛋白经硫酸铵分级沉淀, 再依次经Hiprep 16/10 Q FF阴离子交换柱、Hiload16/60 Superdex 75 凝胶过滤柱和 CM Sepharose FF 阳离子交换柱纯化, 得到电泳纯的包涵体蛋白; 包涵体蛋白经盐酸胍变性溶解、外加血红素重组和柱层析得到了电泳纯的可溶性蛋白. 电喷雾质谱表明, 以这两种形式得到的蛋白分子量相差153.0, 紫外-可见吸收光谱、荧光光谱和圆二色光谱均表明, 这两种形式的蛋白在血红素构象上存在差异, 其热稳定性也不相同.     

人肝中硒蛋白K相互作用蛋白的筛选与验证

以硒蛋白K(SelK)突变体为"诱饵", 采用酵母双杂交系统对人肝cDNA文库进行筛选, 得到一个与SelK相互作用的蛋白──环腺苷酸应答元件结合蛋白3(CREB3). 将SelK与CREB3共同转染酵母细胞, 验证了SelK与CREB3的相互作用; 并采用受体漂白、敏化发射和荧光寿命3种荧光共振能量转移方法进一步验证了二者间的相互作用, 发现其不受SelK中硒代半胱氨酸(Sec)的影响. 推测SelK可能通过其Sec之前的区域与CREB3发生作用, 参与CREB3介导的内质网相关降解过程, 影响相关癌症的转移和发展.     

纺锤体驱动蛋白抑制剂的设计、合成与生物活性研究

纺锤体驱动蛋白(kinesin spindle protein, KSP/Eg5)作为潜在的肿瘤治疗靶点, 使发现KSP抑制剂成为热点. 设计并合成了4-氧基-β-四氢咔啉衍生物作为新型的KSP抑制剂, 并测定了其对KSP的抑制活性, 均优于阳性对照物. 其中化合物9c抑制KSP的IC50＝0.065 μmol•L－1, 优于阳性对照物Monastro l100多倍. 生物活性研究表明为抗肿瘤药物提供了新结构类型的候选化合物.     

Metal Affinity-Based Purification of a Red Fluorescent Protein

A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification should expand the utility of DsRed.     

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E.coil

The entire gene of carboxyltransferase(CT) domain of acetyl-CoA carboxylase(ACCase) from Chinese Spring wheat(CSW) plastid was cloned firstly,and the 2.3 kb gene was inserted into PET28a+ vector and expressed in E.coil in a soluble state.The (His)6 fusion protein was identified by SDS-PAGE and Western blot.The recombinant protein was purified by affinity chromatography,and the calculated molecular mass(Mr) was 88000.The results of the sequence analysis indicate that the cloned gene(GeneBank accession No.EU124675) was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid.The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides,and further to screen new herbicides.     

Cloned s-Lap Gene Coding Area, Expression and Localization of s-Lap／GFP Fusion Protein in Mammal Cells

s-Lap is a new gene sequence from pig retinal pigment epithelial (RPE) cells, which was found and cloned in the early period of apoptosis of RPE cells damaged with visible light. We cloned the coding area sequence of the novel gene of s-Lap and constructed its recombinant eukaryotic plasmid pcDNA3.1-GFP/s-lap with the recombinant DNA technique. The expression and localization of s-lap/GFP fusion protein in CHO and B16 cell lines were studied with the instantaneously transfected pcDNA3.1-GFP/s-lap recombinant plasmid. s-Lap/GFP fusion protein can be expressed in CHO and B16 cells with a high rate expression in the nuclei.     

丝心蛋白基因分子克隆与表达的初步探讨

通过聚合酶链反应扩增丝心蛋白Ｃ亚基结构的基因，并将基克隆到融合蛋白表达载体ｐＲＩＴ２Ｔ质粒中得到ｐＲＩＴ２Ｔ－ＦＬ质粒，在大肠杆菌株Ｐ２３９２内进行表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和免疫印迹反应证明融合蛋白在大肠杆菌中得到了表达。     

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E. coil

The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.     

Expression and Purification of Active Recombinant Human Nerve Growth Factor from Escherichia coli

Introduction Nerve growth factor(NGF) was first discovered and purified by Rita Levi-Montalcini and Stanley Cohen in the 1950s[1,2]. It represents the first cellular growth factor ever discovered and involved in the growth, survival, and differentiation of specific nerve cell popula-tions[3].     

基于Fe3O4@SiO2/Ni-NTA磁性微球的His-tag融合蛋白纯化体系的建立

合成了表面共价结合Ni-氨基三乙酸(Ni-NTA)基团的Fe3O4@ SiO2微球,这种磁性微球可用于分离含有His-tag标签的融合蛋白.微球中心由尺寸约402 nm的Fe3O4微粒组成,赋予了微球极好的磁性分离和离心分离的特性.应用Fe3O4@ SiO2/Ni-NTA磁性微球对含有6×His-tag(6聚组氨酸)标签的蛋白进行了分离纯化,结果表明,10 mg Fe3O4@ SiO2/Ni-NTA微球能够从10mL重组蛋白裂解液中纯化出约1 mg带有6×His-tag标签的融合蛋白.微球的高效分离效果使其能够用于含量较低的带有6×His-tag标签蛋白的分离纯化.     

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

Introduction Matrixmetalloproteinases(MMPs)areafamilyof calciumandzincrequiringendoproteinasesthattogether candegradeallthemaincomponentsoftheextra cellu larmatrixandbasementmembranes[1].MMPsarein volvedinawiderangeofproteolyticevents,innormal andpatholog…