【作者】 张培源；

【导师】 罗贵民；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 单链抗体2F3在大肠杆菌中诱导表达,形成无活性的包涵体。对破碎菌体之后获得的包涵体,以低浓度变性剂进行了初步纯化;使用盐酸胍和巯剂乙醇溶解包涵体,在变性条件下,利用镍离子螯合层析柱纯化,目的蛋白纯度达到电泳一条带。将纯化后的变性蛋白在不同浓度的盐酸胍中平衡,利用360 nm处的光吸收找到单链抗体发生聚集的变性剂浓度;测定荧光发射光谱和圆二色谱,做折叠曲线。其中内源荧光探针为酪氨酸,折叠曲线显示转折点在2 mol/L盐酸胍处,表明酪氨酸所处微环境的改变;外源荧光探针为苯氨基萘磺酸(ANS),曲线折点位于1 mol/L盐酸胍,提示疏水区域的形成;圆二色谱转折点位于2mol/L盐酸胍处,对应二级结构的形成。为了提高单链抗体复性产率,我们利用稀释法进行复性研究,尝试了一系列的小分子添加剂,其中有我们自己合成的两种β-环糊精的衍生物。利用360 nm处的光吸收来反应复性体系中蛋白的聚集程度;通过活力测定来检测正确折叠产物的量。更多还原

【Abstract】 The most important biological macromolecule is protein, whose function isdetermined by its three dimension structure. The structure resolution is necessaryto the understanding of relationship between protein structures and its function,protein de novo design, conformation disease and rational drug design. How thenatural three dimension structure is formed from its amino acid sequence, this isthe protein folding problem.The knowledge of protein folding is powered by the investigation, which gothrough the “self-assembly” and the discovery of the folding assistant molecules.With the advent of post genome period, finding the coding of protein andelucidating structure of protein becomes the main research target;protein massproduction also needs the soluble and bioactive recombinant product. But all theseare enslaved to bottleneck of hetero expression-refolding of inclusion bodies.Inclusion bodies are non-bioactive aggregation of protein with high density, oftenresulting from the high level expression of recombinant protein where the nascentpolypeptide can’t fold properly.The protocol of inclusion bodies refolding includes three parts:denaturalization, purification and refolding. When protein aggregation dissolved,refolding is the polypeptide refolding in vivo: to obtain the folding properly proteinmolecule with bioactivity, denaturant and reducer need to be removed, under theremoval process the molecule refold to its natural conformation. Refoldingefficiency is determined by the relationship between the folding efficiency andaggregating efficiency, protein folding is a first order reaction and aggregating ishigher order reaction, avoidance of the interaction between molecules is extremelyimportant. Means to this is to reduce protein concentration, and to separate theprotein molecule by matrix such as refolding on column.The chaperons and small molecular additives can also be added to therefolding system to get satisfying rate of production with high proteinconcentration. The molecule chaperons need to be removed after refolding andcant be used repeatedly. Enlightened by chaperons some methods which blockshydrophobic regions are developed, such as antibody against the target protein orthe hydrophobic matrix.If the protein has disulfide bonds, to facilitate the formation of the naturalparing, thiol oxidation-reduction pairs are need to accelerate the cysteine exchange.Only the natural disulfide bonds can result in the natural conformation. Commonlyused oxidation reduction system is GSH/GSSG, cysteine/cystine,cysteamine/cystamine, DTT/GSSG and DTE/GSSG. The reduced mercapto isusually 1-3 mmol/l, the proportion of reduced mercapto and oxidated mercaptoranges from 10:1 to 1:1. Moreover, the exchange is favorable in alkalinecircumstance. The protein molecule reaches its thermodynamics stable structuresduring the breakage and formation of the disulfides.Small molecule additives can reduce the aggregation. A series of additiveswas proved to prevent the aggregation. They may:A. stabilize the active state of proteins;B. reduce the stability of wrong folded molecules;C. increase the stability of folding intermediates;D. enhance the stability of unfolded states.Expression of single chain antibody 2F3 in E. coli. results in inclusion bodieswithout bioactivity. After sonication and centrifugation , the pellet was washedwith buffer containing 2 M Urea and solubilized in 6 M Guanidine HCl and 1 mMβ -Mercaptoethanol. The unfolded recombinant protein was purified onIMAC(Immobilized Metal Ion Affinity Chromatography) to the purity of singleband on SDS-PAGE. To recover bio-active proteins, we tried to remove denaturantby dilution the unfolded and reduced scFv into buffer containing small additivesincluding glycerol, sucrose, arginine, β-cyclodextrin and its derivant 6-OTs-β-CD and 2-selenium bridged β-cyclodextrin (2-SeCD).The refolding process wasfollowed by a variety of optical spectroscopy techniques including dynamic andstatic light scattering, intrinsic fluorescence of Trp, extrinsic fluorescence of ANSand circular dichroism.更多还原