人工分子伴侣复性体系辅助溶菌酶复性的研究

研究了人工分子伴侣复性体系中各种因素对溶菌酶复性效果的影响.当缓冲液浓度为23 mmol/L、pH值为8.1、以半胱氨酸为二硫键重排剂时,对人工分子伴侣辅助复性效果较佳.人工分子伴侣体系(CTAB、β-CD和Cysteine)的复性率比只加Cysteine的体系提高约40%,比添加β-CD和Cysteine的体系提高约20%.同时发现当溶菌酶浓度较高时,增加盐酸胍浓度可以提高蛋白质的复性率.     

蛋白质的层析折叠复性

针对包涵体蛋白质复性回收率低,容易凝集的难点,探讨了利用各种层析如：金属鳖合层析,亲和层析,离子交换层析和凝胶过滤层析进行蛋白质折叠复性的原理及应用。在前人工作的基础上发展了一种变性剂浓度梯度凝胶过滤复性蛋白质的新技术。该技术避免了以往凝胶过滤层析复性中变性剂浓度的突然变化。在将其应用于溶菌酶的折叠复性中收到了很好的效果。可以在高蛋白质浓度下得到较高的活性回收率,显示了很大的优越性。为蛋白质层析复性技术的发展提供了新的手段。     

稀释复性法体外复性重组牛凝血酶原-2包涵体

对重组牛凝血酶原-2包涵体的体外重折叠复性进行了研究。凝血酶原-2是α-凝血酶生成过程中的一个最小的单链中间前体。重组牛凝血酶原-2在E.coli中高效表达并形成包涵体,经2.5mol?L?1脲和0.7%Triton X-100洗涤,再用8mol?L?1脲和0.3mol?L?1DTT溶解后,得到了包涵体裂解液。研究优化了体外复性溶液,其组成为含2.7mol?L?1脲、0.6mmol?L?1GSSG、GSSG/GSH0.9、0.1%PEG6000和0.5mol?L?1L-Arg、pH7.4Na3PO4缓冲液。牛凝血酶原-2复性后经透析,再被Echis Carinatus Snake Venom激活,转化为有活性的α-凝血酶,其总活力可达2948U?mL?1。结果表明稀释复性法可用于重组牛凝血酶原-2包涵体的体外重折叠复性。     

反相色谱法研究人粒细胞集落刺激因子的离子交换色谱复性和稀释复性

以反相色谱分析为主要手段,结合活性测定,研究了以包含体形式表达的重组人粒细胞集落刺激因子(rhG-CSF)的稀释复性和离子交换色谱复性. 建立了L-精氨酸离子交换复性蛋白质的方法,将变性、还原的rhG-CSF吸附到高浓度变性剂平衡的离子交换色谱柱上,用L-精氨酸作洗脱剂进行梯度洗脱并同时脱除变性剂,实现了rhG-CSF的复性. 与稀释复性相比,色谱复性的rhG-CSF处于一种中间状态,呈现快速而不同步的动力学特点,这与色谱复性的梯度洗脱及固定相的吸附有关. 同时发现了对复性峰进一步稀释并且温育则可以提高其活性的新现象.     

CTAB辅助溶菌酶复性过程动力学

研究了溶菌酶在十六烷基三甲基溴化铵（CTAB）溶液中的复性过程,通过测定溶液表面张力与酶活力的变化证实变性溶菌酶首先与CTAB形成复合物,进而在氧化-还原剂作用下开始复性并与CTAB发生解离.非还原型SDS-PAGE分析结果表明,复性反应的主要产物有三类：具有天然结构的溶菌酶单体、溶菌酶多聚体及溶菌酶单体与CTAB形成的无活性复合物.不同产物的含量取决于溶液中CTAB与溶菌酶的摩尔比.当变性溶菌酶浓度为0.1～0.4mg•ml^-1,CTAB与溶菌酶摩尔比为5～20时,采用“变性-复性”二态复性模型分析了CTAB辅助溶菌酶复性过程的宏观动力学.结果表明,随CTAB与溶菌酶摩尔比的增大,变性反应的速率常数显著增大,而折叠反应的速率常数先增大后减小,CTAB与溶菌酶的摩尔比为10时,复性率最高.而蛋白质浓度提高则导致折叠反应速率常数减小,变性反应速率常数增大,复性率缓慢下降.     

人工伴侣促进溶菌酶复性动力学

利用复性和聚集竞争反应动力学模型描述自发复性和人工伴侣系统(十六烷基三甲基溴化铵与b -环糊精)促进溶菌酶复性动力学。在酶浓度为0.5~2.0 mgmL-1、盐酸胍浓度为0.5~2.2molL-1范围内,系统分析了盐酸胍浓度和人工伴侣浓度对复性动力学的影响。与自发复性相似,人工伴侣促进溶菌酶复性亦符合3级聚集反应动力学。人工伴侣系统的主要作用是抑制聚集体的生成速率,从而达到提高复性收率的效果。在盐酸胍浓度小于1.2mol稬-1时,人工伴侣系统和盐酸胍对促进变性溶菌酶复性具有协同作用,两者共同作用对促进蛋白质复性具有显著效果。     

表面活性剂辅助重组蛋白质复性

以重组人溶菌酶（rhLys）与重组β-甘露聚糖酶（rMan）为体系,综合采用活性测定、非还原性SDS-PAGE以及荧光发射光谱分析等方法研究了十六烷基三甲基溴化铵（CTAB）、CTAB与β-环糊精（β-CD）组成的人工分子伴侣以及其他种类复性助剂对重组蛋白质复性过程的影响.结果表明CTAB及人工分子伴侣均可有效地辅助rhLys复性,且rhLys与鸡卵清溶菌酶（Lys）呈现出类似的复性过程特性;人工分子伴侣可显著地提高rMan在高浓度下的复性率;表面活性剂带电性质、表面活性剂与蛋白质的摩尔比以及变性蛋白质的浓度等是影响复性率及复性产品分布的主要因素.这些结果对于此类复性技术应用于重组蛋白体系时的工艺选择和优化提供了重要的依据和参考.     

人工伴侣辅助溶菌酶复性的研究

研究了去污剂十门烷基三甲基溴化铵（ＣＴＡＢ）和β－环糊精（β－ＣＤ）联合作用（人工伴侣系统），对变性－还原溶菌酶的复性作用。考察了操作条件对溶菌酶复性收率的影响。结果表明，在人工伴侣系统辅助下，６０ｍｉｎ可使变性溶菌酶的复性收率达８９％，自发复性仅为２７％；并且其最佳复性温度为３７℃，最佳ｐＨ值为７￣９；同时发现，单独使用环糊精也可在一定程度上辅助溶菌酶复性，复性收率为４６％。     

分子伴侣GroEL促进溶菌酶复性动力学

建立了溶菌酶复性的表观动力学模型 ,研究了分子伴侣GroEL促进变性溶菌酶复性的动力学行为 ,包括酶浓度、三磷酸腺苷 (ATP)浓度、GroEL与酶的摩尔比对复性率和复性速率常数的影响 .酶浓度对复性速率常数的影响比对复性率的影响显著 ;酶的复性率和复性速率常数随ATP浓度的增大而提高 ;在蛋白质浓度一定的情况下 ,存在适宜的GroEL和ATP浓度 ,使复性率和复性速率常数最大     

核糖核酸酶A的错流超滤复性

针对重组蛋白高表达形成的包涵体,基于中空纤维膜的错流超滤复性具有较大的应用潜力。为研究不同条件对基于中空纤维膜的错流超滤复性的影响,以核糖核酸酶A (RNase A)为例,以复性过程的活性收率和质量收率作为考察指标,设计了RNase A复性初始浓度(A)、跨膜压力(B)、循环流速(C)的3因素3水平正交实验。结果表明,错流超滤复性过程中,以上复性条件对RNase A的质量收率基本没有影响,对RNase A的活性收率具有显著影响,其较优组合为A1B1C2,即RNase A复性初始浓度为0.3 mg/mL、跨膜压力34.0 kPa、循环流速935 cm/min。在以上复性条件下,RNase A的活性收率可达92.31%,质量收率为77.56%。     

温敏型聚合物PNIPA和P(NIPA-co-SA)的制备及其协助溶菌酶复性研究

采用反相悬浮法合成了温敏型聚合物聚N-异丙基丙烯酰胺(PNIPA)凝胶及其与丙烯酸钠(SA)的共聚凝胶P(NIPA-co-SA),对凝胶的形态、粒径分布、表面特性以及温敏性能进行了考察,共单体丙烯酸钠的引入使得凝胶的最大溶胀倍率和低临界溶解温度(LCST)都有明显的提高。将两种凝胶用于目标蛋白溶菌酶的体外复性过程,考察了其协助复性的效果。结果表明:当蛋白浓度为250μg·mL-1时,加入80mg·mL-1PNIPA凝胶可使溶菌酶的活性回收率由稀释复性的51.3%提高到72.9%;加入120mg·mL-1P(NIPA-co-SA)共聚凝胶可使活性回收率达到71.5%。溶菌酶的浓度越高,与稀释复性相比凝胶协助复性的效果就越好。复性后凝胶可方便地分离回收,PNIPA凝胶重复使用6次后,溶菌酶的活性回收率仍高于稀释复性15%以上。     

Thermosensitive poly(N‐isopropylacrylamide) hydrogel for refolding of recombinant bovine prethrombin‐2 from E. coli inclusion bodies

The poly(N‐isopropylacrylamide) (PNIPA) hydrogel, which is a kind of temperature‐sensitive polymer, was synthesized by inverse suspension polymerization. The microscopy and scan electron microscopy (SEM) of PNIPA hydrogel were studied. The microscope photograph showed that the particles were in the range of 0.2–0.5mm in diameter, with numerous conjoint pores about 1–2μm spreading all over the surface of the beads. The swelling properties of PNIPA gel beads indicated that the lower critical solution temperature (LCST) of the gel was 33°C. The PNIPA prepared was applied to the renaturation of bovine prethrombin‐2 (pThr‐2) from inclusion bodies produced in E. coli. It was observed that PNIPA was quite efficient in assisting protein renaturation at high protein concentration. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the total activity of the thrombin was about 6222U/mL, compared with only 2800U/mL by simple dilution refolding. The kinetics of pThr‐2 refolding with the absence or the presence of PNIPA was also studied respectively. The time required for the refolding with PNIPA gel was a little bit longer than that by the dilution method owing to the diffusion resistance of the protein into the network of the gel and the hydrophobic interaction between the protein and the polymer. The mechanism of the enhancement for the PNIPA gel to the refolding was further discussed. The porosity of the PNIPA hydrogel allows penetration of the unfolded protein into the inside of the polymer with a hydrophobic side chain, which can facilitate the formation of intermediate via hydrophobic interaction with the unfolded protein and the folding intermediate that are liable to re‐aggregation. About 1.2mg of purified active thrombin could be recovered from 1 L of cells, which greatly facilitated the scale‐up to the quantities of protein necessary for further functional and structural studies. A novel protein renaturation method mediated by PNIPA hydrogel beads, which highly increases the refolding efficiency with easy handling, recycling, and low cost, was proposed. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1734–1740, 2005     

Structure and protein separation efficiency of poly(N-isopropylacrylamide) gels: Effect of synthesis conditions

Crosslinked poly(N-isopropylacrylamide) (PNIPA) gels with different crosslink densities in the form of rods and beads were prepared by free-radical crosslinking copolymerization. Solution and inverse suspension polymerization techniques were used for the gel synthesis. The gels were utilized to concentrate dilute aqueous solutions of penicillin G acylase (PGA), bovine serum albumin (BSA), and 6-aminopenicillanic acid (6-APA). The discontinuous volume transition at 34°C observed in the gel swelling was used as the basis of concentrating dilute aqueous protein solutions. PNIPA gels formed below 18°C were homogeneous, whereas those formed at higher temperatures exhibited heterogeneous structures. The water absorption capacity of PNIPA gels in the form of beads was much higher, and their rate of swelling was much faster than the rod-shaped PNIPA gels. It was also found that the polymerization techniques used significantly affect the properties of PNIPA gels. The separation efficiency decreased when the protein molecules PGA or BSA in the external solution were replaced with small-molecular-weight compounds, such as 6-APA. The protein separation efficiency by the gel beads increased to 100% after coating the bead surfaces with BSA. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67: 805–814, 1998     

胆固醇配基色谱在蛋白质复性中的应用

以三聚氯氰为连接臂,采用两步法将疏水配基胆固醇修饰到交联琼脂糖（Sepharose）介质上,构建了胆固醇配基色谱。以溶菌酶（Lys）为模型蛋白,考察了胆固醇配基色谱的复性效果,并研究了流速、上样质量浓度和上样量对色谱复性效果的影响,确定了溶菌酶复性的最佳方法。研究结果表明,当溶菌酶上样量为1.00 mL,介质含0.25 mg溶菌酶时,在0.02 mL/min操作流速下进行吸附-洗脱复性,其最终蛋白收率和活性收率分别为96.3%和91.4%,有效促进了溶菌酶的复性。     

The effect of preparation temperature on the swelling behavior of poly (N-isopropylacrylamide) gels

Summary The role of the preparation temperature of poly(N-isopropylacrylamide) (PNIPA) gels on their swelling behavior in water and in aqueous solutions of sodium dodecylbenzenesulfonate was investigated. PNIPA gels were prepared by free-radical crosslinking copolymerization of N-isopropylacrylamide and N,N'-methylene(bis) acrylamide in water at fixed monomer and crosslinker concentrations. The equilibrium swelling ratio of the gels increases first slightly up to about 20°C then rapidly with increasing gel preparation temperature. Magnitude of the collapse transition in water at 34°C becomes larger as the gel preparation temperature increases. Calculations indicate a decrease in the effective crosslink density of PNIPA gels with increasing preparation temperature. The gels prepared at temperatures higher then 20°C were heterogeneous consisting of highly crosslinked regions interconnected by the PNIPA chains. Received: 3 May 2000/Revised version: 3 July 2000/Accepted: 17 July 2000     

Effect of the SA content of a novel thermo‐sensitive P(NIPAM‐co‐SA) copolymer on denatured lysozyme refolding in vitro

A novel hydrogel of P(NIPAM‐co‐SA) copolymer was synthesized by inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of poly(N‐isopropylacrylamide) (PNIPAM). The morphologies, size distribution and thermosensitive characteristics of gel particles were studied and the maximal swelling ratio and LCST (Lower Critical Solution Temperature) of gel particles increased obviously with the addition of SA comonomer. When the protein concentration was 250 μg/mL, the optimized refolding conditions of denatured lysozyme with P(NIPAM‐co‐SA) hydrogel were that operating at the temperature of 35°C and a urea concentration of 2M, in which the mass ratio of P(NIPAM‐co‐SA) hydrogel with 4% SA copolymerized to lysozyme was 10 : 1. Under the optimized conditions, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM‐co‐SA) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA comonomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein molecules in the course of lysozyme refolding compared with the presence of PNIPAM polymer. All results above demonstrate that the P(NIPAM‐co‐SA) is a cost effective additive with tunable hydrophobicity for application in the refolding of recombinant proteins expressed as inclusion bodies in vitro. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Mono-sized microspheres modified with poly(ethylenimine) facilitate the refolding of like-charged lysozyme

Three mono-sized poly(glycidyl methacrylate) (PGMA) microspheres of 0.8, 1.8 and 2.5 μm in particle sizes were prepared via dispersion polymerization. Poly(ethylenimine)s (PEIs) of different molecular weights (60 000 and 1200) were coupled to the PGMA bead surface to prepare cationic beads of different charge densities and ligand (cationic group) structures. The cationic microspheres were used to explore the effect of solid phase properties on like-charged lysozyme refolding. The refolding yield increased with increasing bead concentration and charge density. At low bead concentration range, it increased more significantly with bead concentration with the microspheres of higher charge density. This indicates that the microspheres of high charge density are beneficial in facilitating protein refolding. The refolding yield was independent of the cationic group structure. However, PEI of higher molecular weight was favorable in the preparation of microspheres of higher charge density. Smaller-sized particles have higher specific surface area, so they facilitated lysozyme refolding more significantly. By addition of the charged beads, about 90% refolding yield of 4 mg/mL lysozyme could be achieved. The studies provided more insight into the effects of like-charged solids on protein refolding, which would help design more efficient charged media for facilitated protein refolding applications.     

Refolding of lysozyme at high concentration in batch and fedbatch operation

Based on optimization of denaturing conditions and the character of time course of protein refolding, renaturation by dilution in batch and fed-batch operation to improve yield as well as the initial and final protein concentrations has been studied. The optimum DTT in denatured solution was 30 mM. Urea can suppress protein aggregation to sustain pathway of correct refolding at high protein concentration. Fed-batch operation was better than batch dilution with comparison of yield recovery in large-scale downstream processes. Under our research condition in fed-batch operation, lysozyme was successfully refolded from initial protein concentration of up to 40 mg/mL by dilution 20-fold, the yield recovery was nearly 60%.     

高浓度变性-还原溶菌酶的流加复性动力学特性

研究了高浓度变性-还原溶菌酶的流加复性过程动力学特性,重点考察了影响复性速率和复性收率的主要因素,包括盐酸胍浓度、酶浓度和氧化还原剂浓度.结果表明,与直接稀释复性相比,流加操作可有效降低肽链分子间聚集,提高蛋白质的复性收率;在流加复性条件下,随着酶浓度的提高,复性速率和复性收率均有所下降,但适当提高复性液中盐酸胍浓度仍可获得高浓度蛋白质的高复性收率.另外,随着变性酶浓度的提高,需要适当提高复性液中氧化型谷胱甘肽浓度,以加快溶菌酶分子内二硫键的形成,提高复性反应速度.     

重组人成骨蛋白-1复性条件的优化

目的优化重组人成骨蛋白-1(rhOP-1)包涵体蛋白的复性条件。方法将表达rhOP-1的大肠杆菌菌体在冰浴下超声裂解,分离提取包涵体,用8mol/L尿素溶解,纯化后进行梯度透析复性。利用TotalLab软件分析目的蛋白二聚体的含量,用体内法和体外法测定其生物学活性。结果经纯化后,目的蛋白纯度达97%以上。最佳复性条件为4℃,pH9.0,蛋白浓度为0.4～0.8mg/ml,尿素浓度为2mol/L,L-Arg浓度为0.4mol/L;目的蛋白复性率达75%以上,复性后蛋白具有较高的生物学活性。结论已确定了rhOP-1包涵体梯度透析复性的最佳条件。