重组人胰激肽原酶复性工艺的研究

目的:研究重组人胰激肽原酶包涵体变性及复性的工艺。方法:对本实验室构建的重组人胰激肽原酶大肠杆菌进行IPTG诱导表达表达成功后,菌体经超声破碎释放包涵体,包涵体经洗涤、变性、稀释和尿素梯度凝胶过滤色谱这两种方法复性后(Sephadex-G75),通过测定酶活检验复性效果。结果:①重组人胰激肽原酶工程菌经过IPTG诱导后能够表达目的蛋白,目的蛋白以包涵体形式存在,将细胞破碎后,包涵体经过3次洗涤,纯度达到71.93%;②变性包涵体经24小时稀释复性后,蛋白浓度达到72.61μg/m L,酶的比活达到13.84 U/mg;③变性包涵体经过2个小时的尿素梯度凝胶过滤复性后,蛋白浓度可达到830.07μg/mL,酶的比活达到48.61 U/mg。结论:两种复性方法均可以使包涵体达到一定的浓度和比活,比较发现尿素梯度凝胶过滤色谱具有复性时间短和比活力高等优点,可作为重组人胰激肽原酶复性的一种有效的手段。     

蛋白质的排阻色谱复性的新进展

外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。     

人血小板衍生生长因子BB亚型包涵体复性与纯化

目的:优化人血小板衍生生长因子BB亚型(PGDF-BB)包涵体复性方法与纯化条件,获得具有较好生物活性的重组PGDF-BB蛋白。方法:对PGDF-BB包涵体以梯度尿素进行变性,选择最佳包涵体变性浓度;比较不同复性条件下的复性率,稳定PGDF-BB包涵体复性方法;参照该蛋白的理化性质,选择适合PGDF-BB重组蛋白的纯化方法。结果:原核系统内实现了PGDF-BB的高表达;通过优化包涵体复性方法,重组蛋白的包涵体复性率可达40%以上;经过多个纯化方法相结合,PGDF-BB的纯度达到95%。结论:通过实验条件的优化,提高了PGDF-BB包涵体复性率,获得高纯度、高生物活性的重组PGDF-BB蛋白。     

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 。     

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。     

人血小板衍生生长因子BB的原核高表达及其包涵体复性

目的:在原核系统内获得高表达的人血小板衍生生长因子BB(PGDF-BB),并对形成的包涵体进行复性。方法:对PGDF-BB核酸编码序列进行优化,构建pET-22b-PGDF-BB表达载体,以提高PCDF-BB的表达量;优化PGDF-BB包涵体复性条件,提高蛋白复性率和生物活性。结果:构建了pET-22b-PGDF-BB高效表达载体,原核表达的重组人PGDF-BB占细菌总蛋白的25%,PGDF-BB包涵体复性率达到15%。结论:对表达序列的优化设计可显著提高蛋白的表达量,复性方法的改良提高了蛋白的复性率和生物活性。     

内生真菌Shiraiasp.Slf 14中菊粉酶基因的克隆、表达及包涵体复性

为了实现内生真菌Shiraia sp.Slf 14菊粉酶基因在大肠杆菌中的高效表达,建立有效的包涵体复性技术,获得有活性的重组菊粉酶,本研究通过提取Shiraia sp.Slf 14的总RNA,反转录合成cDNA,设计PCR引物扩增出菊粉酶基因,将其克隆至pET-22b(+)载体后转入E.coli BL21(DE3),利用SDS-PAGE法检测IPTG诱导表达后重组蛋白的表达情况,并进一步检测了包涵体复性及重组酶酶活情况,最终成功获得了相对分子量为62.07 kD的重组蛋白,成功复性包涵体,复性率为25.23%,重组菊粉酶活力为6.84 U/m L。本研究为活性重组菊粉酶的获得及包涵体复性提供了新的方法和依据。     

重组包涵体蛋白质的折叠复性

综述了减少包涵体形成、包涵体分离和溶解以及包涵体折叠复性的策略及其最新进展 .详细讨论了包涵体蛋白质折叠复性的基本原则、包涵体折叠复性促进剂和包涵体折叠复性方法     

一种从大肠杆菌包涵体中分离纯化GST-TRAF6融合蛋白的方法

利用8 mol/L尿素溶液对表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白进行变性,通过逐级稀释复性的方法对尿素溶解后的GST-TRAF6融合蛋白进行复性,将复性后的GST-TRAF6融合蛋白进一步利用谷胱甘肽琼脂糖树脂亲和层析的方法进行分离纯化,将分离纯化后的蛋白通过Western blot方法进行验证,最后利用体外泛素化反应检测经包涵体变性、复性和纯化后的GST-TRAF6融合蛋白的生物学活性。经过包涵体变性、梯度稀释复性和谷胱甘肽琼脂糖树脂亲和层析3个步骤后纯化得到纯度达90%以上、浓度为396 ng/μL的蛋白质溶液。利用GST蛋白作为对照,经Western blot验证表明,纯化得到的蛋白确为GSTTRAF6融合蛋白。进一步利用体外泛素化反应分析其泛素连接酶活性发现,17 ng/μL浓度的GST-TRAF6融合蛋白能够以泛素分子作为底物在5 min内快速催化自由泛素链的生成。结果表明,表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白经尿素变性溶解后能够成功复性并分离纯化,在溶解性改变的同时恢复了其泛素连接酶活性。为从大肠杆菌包涵体中大规模分离纯化蛋白质提供了一种新的复性方法。     

重组蛋白ApSerpin-FA72的包涵体复性及活性研究

[目的]原核表达及制备重组光滑鳖甲丝氨酸蛋白酶抑制剂(Ap Serpin-FA72),探索包涵体最优复性条件与最适酶反应条件。[方法]采用超声破碎获得大量包涵体,通过包涵体的洗涤、包涵体的溶解方法对包涵体进行纯化,获得高纯度的包涵体进行复性液成分与复性方法的摸索。测定Trx A-Ap Serpin-FA72对胰蛋白酶的IC50、最适反应p H和最适反应温度。[结果]在32℃、180 r/min、0. 4 mmol/L IPTG浓度下以沉淀形式表达大量蛋白,通过包涵体复性在含有L-精氨酸复性液中获得有生物活性的Trx A-Ap Serpin-FA72,对胰蛋白酶的IC50为0. 48μmol/L,p H在7～9,温度在60℃时有较高的抑制活性。[结论]L-精氨酸是复性液中重要的组成部分,复性的重组蛋白Trx A-Ap SerpinFA72对胰蛋白酶有较强的抑制能力,是一种热稳定较好的弱碱性胰蛋白酶抑制剂。     

Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins

Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.     

Practical considerations in refolding proteins from inclusion bodies

Refolding of proteins from inclusion bodies is affected by several factors, including solubilization of inclusion bodies by denaturants, removal of the denaturant, and assistance of refolding by small molecule additives. We will review key parameters associated with (1) conformation of the protein solubilized from inclusion bodies, (2) change in conformation and flexibility or solubility of proteins during refolding upon reduction of denaturant concentration, and (3) the effect of small molecule additives on refolding and aggregation of the proteins.     

Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography

The presence of inclusion body impurities can affect the refolding yield of recombinant proteins, thus there is a need to purify inclusion bodies prior to refolding. We have compared centrifugation and membrane filtration for the washing and recovery of inclusion bodies of recombinant hen egg white lysozyme (rHEWL). It was found that the most significant purification occurred during the removal of cell debris. Moderate improvements in purity were subsequently obtained by washing using EDTA, moderate urea solutions and Triton X-100. Centrifugation between each wash step gave a purer product with a higher rHEWL yield. With microfiltration, use of a 0.45 micron membrane gave higher solvent fluxes, purer inclusion bodies and greater protein yield as compared with a 0.1 micron membrane. Significant flux decline was observed for both membranes. Second, we studied the refolding of rHEWL. Refolding from an initial concentration of 1.5 mg ml-1, by 100-fold batch dilution gave a 43% recovery of specific activity. Purified inclusion bodies gave rise to higher refolding yields, and negligible activity was observed after refolding partially purified material. Refolding rHEWL with a size exclusion chromatography based process gave rise to a refolding yield of 35% that corresponded to a 20-fold dilution.     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

SOn-column Protein Refolding for Crystallization

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.     

Refolding of protein inclusion bodies directly from E. coli homogenate using expanded bed adsorption chromatography

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method

Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial‐and‐error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix‐assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion‐exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α‐lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix‐assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used.     

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25 column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding. Revisions requested 31 October 2005; Revisions received 20 December 2005