脯氨酰顺—反异构酶的纯化及对重组蛋白质折叠反应…

脯氨酰异构是蛋白质折叠反应的限速步骤之一,体内被脯氨酰顺－反异构酶（ＰＰＩ）所催化。为了研究ＰＰＩ在重组蛋白体外折叠复性中的作用,我们自猪肾脏中纯化了ＰＰＩ,并对重组蛋白的酶促折叠过程进行了探讨。结果表明,ＰＰＩ催化的重组蛋白的折叠率和比活性,ＰＰＩ催化的重组蛋白的折叠反应主要是提高了它们了折叠速率,而不增加正确折叠率的比活性。ＰＰＩ在很低的浓度下即有很高的催化活性。     

包含体的体外复性策略

外源基因在大肠杆菌中高水平表达时,通常会形成不溶性、无活性的蛋白聚集体即包含体。包含体富含表达的重组蛋白,经分离、变性溶解后须再经过一个合适的复性过程实现变性蛋白的重折叠,才能够得到生物活性蛋白。近年来,发展了许多特异的策略和方法来从包含体中复性重组蛋白。介绍稀释法、透析及分子排阻、固定化金属离子亲和层析、疏水层析复性等策略和进展;物理化学因素、前导肽协助蛋白折叠;人工分子伴侣辅助蛋白折叠及反胶束、多聚物用于蛋白复性。     

脯氨酰顺－反异构酶的纯化及对重组蛋白质折叠反应的催化性能

脯氨酰异构是蛋白质折叠反应的限速步骤之一,体内被脯氨酰顺－反异构酶(PPI)所催化．为了研究PPI在重组蛋白体外折叠复性中的作用,我们自猪肾脏中纯化了PPI,并对重组蛋白的酶促折叠过程进行了探讨．结果表明,PPI催化的重组蛋白的折叠反应主要是提高了它们的折叠速率,而不增加正确折叠率和比活性,PPI在很低的浓度下即有很高的催化活性．     

SARS冠状病毒未知蛋白Orf 9b的克隆、表达和结构分析

从SARS病毒基因组中克隆Orf 9b基因并构建pET32-Orf 9b原核表达质粒,在原核细胞中表达目的重组蛋白并将其纯化,经肠激酶酶切后,得到分子量为11 kD的Orf 9b蛋白。ELISA检测表明重组Orf 9b蛋白可以与SARS康复病人血清反应而不与健康人血清反应。用圆二色光谱和红外光谱初步分析了重组Orf 9b蛋白的二级结构组成。圆二色光谱显示重组Orf 9b蛋白中α-螺旋占12.5%,β-折叠占40%,无规则卷曲占47.5%,红外光谱显示重组Orf 9b蛋白中α-螺旋占13.7%,β-折叠占47.5%,无规则卷曲占37.9%,两种检测方法对重组Orf 9b蛋白结构分析的结果基本一致,提示Orf 9b蛋白富含β折叠,而α-螺旋含量较少。获得该重组蛋白及对其结构的初步分析为进一步研究Orf 9b蛋白的功能奠定基础。     

包涵体蛋白复性的几种方法

外源基因在大肠杆菌中高水平表达时,通常会形成无活性的蛋白聚集体即包涵体。包涵体富含表达的重组蛋白,经分离、变性溶解后须再经过一个合适的复性过程实现变性蛋白的重折叠,才能够得到生物活性蛋白。     

包涵体蛋白体外复性的研究进展

外源基因在大肠杆菌中高水平表达时 ,通常会形成无活性的蛋白聚集体即包涵体。包涵体富含表达的重组蛋白 ,经分离、变性溶解后须再经过一个合适的复性过程实现变性蛋白的重折叠 ,才能够得到生物活性蛋白。近年来 ,发展了许多特异的策略和方法来从包涵体中复性重组蛋白。最近的进展包括固定化复性以及用一些低分子量的添加剂等来减少复性过程中蛋白质的聚集 ,提高活性蛋白的产率。     

BACE蛋白的表达、纯化和活性测定

在大肠杆菌中表达、纯化并重新折叠以获得有活性的酸性蛋白水解酶 (BACE蛋白 )———一种与阿尔茨海默病 (AD)发病相关的蛋白水解酶。克隆BACE活性区的表达序列到原核表达载体 pET11a中 ,经E .coliBL2 1(DE3)表达 ,从包涵体中获取蛋白质 ,电泳鉴定后经梯度反向快速折叠法重新折叠 ,柱层析分离纯化 ,得到了表达的重组可溶性BACE蛋白 ;用高效液相色谱、质谱等方法检测其对人工合成多肽底物的水解作用 ;测定了BACE蛋白的酶促动力学常数。结果表明 ,得到的重组BACE蛋白具有水解人工合成小肽底物的活性。     

包涵体复性研究进展(英文)

用基因工程技术在大肠杆菌高水平表达重组蛋白时,通常形成无生物活性的包涵体。包涵体在体外经分离、溶解与重折叠后可实现复性,表现为具有生物活性的蛋白。总结了包涵体的相关复性技术,重点介绍重折叠的最新进展情况 。     

重组蛋白在大肠杆菌周间腔的分泌表达

大肠杆菌是用于生产重组蛋白的重要工程宿主菌。但是,要获得足够的正确折叠的蛋白还存在一定的缺陷,其中一种解决此问题的方法就是使重组蛋白分泌到大肠杆菌的周间腔里。在这篇综述中,主要讨论了使重组蛋白分泌表达至大肠杆菌周间腔的近期的研究进展。     

共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

为了提高甘露聚糖酶ManA在毕赤酵母中分泌表达的酶活,选择毕赤酵母内质网未折叠蛋白反应(Unfolded protein response,UPR)激活调控因子HAC1与5种毕赤酵母蛋白折叠相关的分子伴侣ERO1、PDI、PDI1、CPR5、BiP,通过构建pPICZA-HAC1等6种胞内表达重组质粒,分别电转化至分泌表达ManA的毕赤酵母重组菌中胞内共表达,并分析其重组菌摇瓶发酵时ManA表达的影响。结果发现在摇瓶发酵水平,胞内共表达HAC1、ERO1、PDI的重组菌发酵上清液中的ManA酶活力分别提高了26%、15%、20%,其重组菌发酵上清液的酶活力分别达到1 014 U/mL、925 U/mL、965 U/mL。通过对各重组菌上清液酶活力、胞内滞留酶活力、上清液蛋白浓度数据进行分析,进一步选择将HAC1、ERO1、PDI进行两基因或三基因组合,并分别在分泌表达ManA的重组菌胞内共表达,但各共表达重组菌发酵上清液的酶活力都没有进一步的提升。单独共表达HAC1或者分子伴侣ERO1、PDI可以辅助ManA的正确折叠,提高其蛋白表达。     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

耐冷希瓦氏菌外膜蛋白的体外折叠研究

目的:旨在建立耐低温革兰氏阴性菌外膜蛋白体外折叠体系,为膜蛋白合成耐低温机制提供理论基础。方法:以包涵体的形式在大肠杆菌中过量表达了来源于耐低温希瓦氏菌的OmpA同源外膜蛋白Omp74的全蛋白质和N端跨膜结构域,纯化包涵体后,用高浓度尿素或强阴离子表面活性剂溶液溶解包涵体,以非离子表面活性剂为折叠介质,建立该外膜蛋白的体外折叠体系,同时以大肠杆菌的OmpA作为对照进行了比较研究。结果:与OmpA相比,Omp74体外折叠受温度影响较小,低浓度的阴离子表面活性剂能促Omp74的折叠,但对OmpA的折叠没有影响;C端结构域抑制Omp74在表面活性剂中的折叠;Omp74在0.5%的月桂酰基麦芽糖苷(DDM)和0.4%的十二烷基肌氨酸钠的混合溶液中能达到接近100%的折叠效率。     

重组白细胞介素2体外折叠的实验研究

包涵体中的重组蛋白抽提后可以在变性状态下纯化,而纯化后的体外折叠（即复性）是基因工程下游处埋中的重要环节。荧光光谱研究表明,ＩＬ－２分子折叠过程中荧光强度逐渐减小,最大发射峰由３１６ｎｍ红移到３４８ｎｍ。以Ｔｒｐ残基的暴露程度反映分子的折叠状态。ＧＭ－ＣＳＦ在折叠过程中的荧光强度有类似变化;凝胶排阻ＨＰＬＣ可以检测折叠过程中的聚合体;而反相ＨＰＬＣ可以将ＩＬ－２分成三个相互独立的异构体色谱峰。据此可以计算出ＩＬ－２分子的正确折叠率。常用的稀释复性方法,随着ＩＬ－２浓度的增高,它的正确折叠率逐渐降低,蛋白浓度的对数与正确折叠率之间大致呈线性关系。当ＩＬ－２浓度为１ｍｇ／ｍＬ时,其正确折叠率仅为３０％,而采取较低的蛋白浓度进行复性会因大量的样品体积导致后期纯化的困难。     

Oxidative refolding of lysozyme assisted by DsbA, DsbC and the GroEL apical domain immobilized in cellulose

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBD_Cex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r² > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k _N) and aggregation (k _A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.     

Optimizing protein folding to the native state in bacteria.

A correctly folded protein is usually both active and soluble. This review focuses on novel ways to improve the folding of recombinant proteins during production in bacteria and includes a few tips for refolding proteins. Major results in correlating protein primary structure with proper folding and stability, and the production of viral antigens and antibodies in bacteria are also discussed.     

A first-order-like state transition for recombinant protein folding

Normally, proteins will aggregate and precipitate by direct folding processes. In this study, we report that quasi-static processes can restore both the structure and bio-function of two kinds of fish recombinant growth hormones (Plecoglossus altivelis and Epinephelus awoara). The conformational changes and the particle-size-distribution (PSD) of each refolding intermediate can be monitored by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS), respectively. Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions of the proteins were restored during the last two refolding stages, as elucidated by thermal stability tests. This is consistent with a sequential model. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrank to their native-like sizes after the first refolding stage. This is consistent with a collapse model. After comparison with the data on the direct folding process, it is concluded that the denaturant-containing protein folding reaction is a first-order-like state transition process.     

Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled?

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Kinetic intermediate in the folding of human prion protein

Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.