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。     

蛋白质在大肠杆菌间周质中的折叠

随着分子伴侣的发现和外源基因表达技术的发展 ,大肠杆菌间周质蛋白质的折叠成为研究热点。间周质 (ｐｅｒｉｐｌａｓｍ)是革兰氏阴性菌内膜与外膜之间的区域 ,对外界环境变化的适当能力很脆弱 ,例如ｐＨ、温度、渗透压[1] 。重组技术表达的重要蛋白质大多含有二硫键 ,二硫键的形成在真核生物是在内质网中完成的 ,而大肠杆菌是在间周质中进行的。催化大肠杆菌间周质蛋白质折叠会遇到两个限速步骤 ,被两类酶催化 :二硫键氧化还原酶 (Ｄｓｂ)和肽酰 脯氨酰顺反异构酶 (ｐｅｐｔｉｄｙｌ ｐｒｏｌｙｌｃｉｓ ｔｒａｎｓｉｓｏ ｍｅｒａｓｅ ,…     

Pin1与β-链蛋白——Wnt信号途径的致肿瘤机制

β-链蛋白（β—catenin）是Wnt信号途径核心成员。Wnt信号传人细胞核致β-链蛋白磷酸化以后,磷酸化依赖肽酰脯氨酰异构酶（Pin1）特异性地催化β-链蛋白（p）丝／苏氨酰-脯氨酰肽键的顺反异构[反式（trans）到顺式（cis）],结果β-链蛋白结构稳定、保持活性状态。Pin1机制是Wnt信号途径致肿瘤重要新机制,并与途径信号分子基因突变等完全无关。     

小球藻亲环蛋白A的酶活性及过表达拟南芥抗盐性分析

在前期研究中分离到噬碳酸盐小球藻（Chlorella sp.X1）的亲环蛋白基因CsCyp1A（登录号：KY207381）,编码CsCyp1A蛋白是否具有肽酰脯氨酰顺反异构酶（PPIase）活性功能是进一步研究它参与小球藻耐盐生物学过程的基础。通过His标签的pQE-30-CsCyp1A原核蛋白表达载体,经IPTG诱导它的E.coli M15菌株表达融合蛋白,Nitra-柱纯化后获得纯化的30 kDa左右的His-CsCyp1A融合蛋白质,Western杂交检测到HisCsCyp1A蛋白的杂交信号。酶活性分析显示,HisCsCyp1A融合蛋白中生色基团的产生速度明显快于对照,表明CsCyp1A蛋白可以催化底物N-succinyl-Xaa-Pro-Phe-p-nitroanilide中Xaa-Pro肽键的顺反折叠,说明纯化的CsCyp1A蛋白具有PPIase活性。典型的亲环蛋白家族成员具有肽酰脯氨酰顺反异构酶（PPIase）活性,参与折叠和转运、信号转导、免疫调节、细胞凋亡等生物学过程。真空渗入法侵染转化的拟南芥在35S启动子驱动下超表达CsCyp1A基因,耐盐性研究表明它提高了过表达株系对NaCl胁迫的耐性,揭示小球藻CsCyp1A基因的抗盐性功能,它将成为抗逆育种的基因资源。本研究也为探索小球藻亲环蛋白A的抗盐碱胁迫生物学作用和分子育种奠定了基础。     

亲环蛋白的结构、细胞定位和生物学功能

亲环蛋白(cyclophilin, CyP)家族是一类广泛存在于原核和真核生物体内, 并在结构上高度保守的多功能蛋白质.该家族属于具有催化含脯氨酸的寡肽底物顺反异构作用的肽脯氨酰顺反异构酶(peptidyl-prolyl cis-trans isomerases, PPIase).CyP通过催化含肽脯氨酰的顺反异构而帮助蛋白质折叠和组装.此外, CyP还起着分子伴侣的作用, 在应激反应中参与调节信号转导途径, 并影响RNA的剪接过程.自最初CyP作为免疫抑制剂环孢素A (cyclosporin A , CsA)的细胞受体被发现以来, 目前大约有130种CyP同源异构体被发现和克隆.CyP家族各成员间不仅分子量不同, 而且在细胞分布及功能上也存在着差异.现从CyP的结构、细胞定位及其功能等3个方面对其作一阐述.     

亲环蛋白在病毒感染中的作用

亲环蛋白家族是一类具有肽酰-脯氨酰顺反异构酶活性的蛋白.亲环蛋白不仅具有帮助蛋白质组装和折叠、参与调节细胞内信号转导通路等多种生物学功能,还在病毒感染及病毒的复制周期中起到重要作用.本文就亲环蛋白在病毒感染过程中的具体分子机制进行综述,旨在更深入的阐明病毒的感染过程并为寻找潜在的抗病毒药物靶标提供新的理论依据及思路.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

二硫键异构酶的纯化及其对重组蛋白体外折叠的影响

自牛肝中纯化了蛋白二硫键异构酶(PDI),并对重组蛋白的酶促折叠过程进行了探讨．结果表明。在等摩尔PDl的催化作用下,可使1mg／ml的IIJ-2的正确折叠率提高到58%以上,比活性由4×10⁶u／mg增加到8．2×10⁶u／mg,PDl还能部分纠正二硫键错配的IL-2异构体成为正确折叠的lL-2和防止IL-2通过cys的链问交联形成聚合体。GM-CSF在PDl催化下也有类似的结果。PDl作用的关键是它所催化的琉基一二硫键的交换反应。     

脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析

Cyclophilin A(简称CypA)由PPIA(Peptidylprolyl isomerase A)基因编码,是典型的Cyclophilin家族蛋白,具有肽基脯氨酰顺反异构酶活性,在蛋白质的折叠和转运、信号转导、炎症、免疫调节、细胞凋亡及病毒复制等生物学过程中发挥着重要作用。本研究重点探讨了不同脊椎动物CypA的肽基脯氨酰顺反异构酶活性及其遗传变异。根据Gen Bank数据库PPIA基因的序列信息,克隆了脊椎动物的PPIA基因并构建其原核表达载体,其中鼠耳蝠(Myotis davidi)和绿头鸭(Anas platyrhynchos)的PPIA基因序列为首次报道。利用大肠杆菌表达GST-Cyp A融合蛋白并进行亲和层析,切除GST标签后再进行分子筛层析,获得纯化的CypA蛋白。利用胰糜蛋白酶偶联法测定CypA的肽基脯氨酰顺反异构酶活性,发现12种脊椎动物CypA的肽基脯氨酰顺反异构酶活性无显著差异。同时,通过一系列遗传变异和分子进化分析,发现12种脊椎动物CypA的酶活位点、CsA结合位点等重要功能域的氨基酸序列完全一致,而且其结构和在染色体中的基因定位也非常保守。研究结果表明,脊椎动物CypA的关键功能域高度保守,这是CypA维持其酶活及生物学功能的有力保障。     

烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板,反转录合成cDNA;再以cDNA为模板,通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因,构建表达载体pPIC9K-PEP;电转化酵母宿主菌Pichia pastoris GS115,获得重组菌PEP-09;纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65°C,有较好的温度稳定性,在55°C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5,在pH 3.0 9.0范围内有很好稳定性,在pH 6.0 8.0的缓冲液中37°C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达,重组酶活性稳定,有一定的应用潜力。     

Prolyl isomerases catalyze antibody folding in vitro.

Some slow-folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl-peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate-limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of tunicamycin-inducible gene A polypeptide from Aspergillus niger.

Tunicamycin-inducible gene A polypeptide (TIGA) is a member of the protein disulfide isomerase (PDI) family and is suggested to facilitate the folding of nascent polypeptides. The functional properties of TIGA were investigated here. TIGA acted as an isomerase, catalyzing the refolding of denatured and reduced ribonuclease A. TIGA also exhibited chaperone activity in the refolding of denatured prochymosin but not in the refolding of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), indicating that it had substrate specificity with respect to chaperone activity. Detailed study with a series of thioredoxin-motif (trx-motif) mutants revealed that the 2 trx-motifs of TIGA were not equal in activity. The N-terminal trx-motif was more active than the C-terminal trx-motif, and the first cysteine in each trx-motif was necessary for isomerase activity.     

Energetics of protein structure and folding

The available experimental date on the kinetics of unfolding and refolding of small proteins are reviewed. Excluding slow transitions in the unfolded protein due to cis–trans isomerization of peptide bonds, the rate-limiting transition state in both unfolding and refolding is concluded to be a high-energy distortion of the fully folded state. Partially folded intermediates are undoubtedly important for folding, but their formation is normally not rate limiting. A simple model is used to illustrate some of the aspects of protein-folding energetics.     

大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A（Disulfide bond formation protein A,DsbA）是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys³⁰残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys³⁰残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli

Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis‐folding, loss of protein, and high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing protein folding‐strong anion exchange chromatography (PF‐SAX). The purification method is also standardized for optimal concentration of solution additives, pH, and mobile phase composition. The results showed purification of rhASP with anion exchange chromatography was effective. Phosphate buffer and slightly alkaline pH produced significant recovery yields and purity profiles. The effect of solution additives such as arginine, glycerol, TMAO, sorbitol, dextran, glutamate, and fructose on rhASP renaturation is also investigated. Significant results were achieved using arginine‐TMAO combination in terms of purity, recovery yield and specific activity of 99%, 78%, and 210 IU/mg, respectively. The work concludes that PF‐SAX refolding method is superior to other conventional methods and it can be applied to large scale purification of rhASP produced in E. coli. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1036–1044, 2018     

Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase

A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (T_m) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and T_m values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

     

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.