人工伴侣与凝胶过滤色谱耦合作用促进高浓度溶菌酶复性研究

研究了人工伴侣系统,即十六烷基三甲基溴化铵(CTAB)和β 环糊精(β CD)促进高浓度变性 还原溶菌酶的氧化复性。确定CTAB与β CD的浓度比为1∶4,CTAB与变性溶菌酶的结合时间在10—50min内复性收率较高。同时发现,利用人工伴侣和凝胶过滤色谱的耦合作用可有效提高溶菌酶的复性收率,在连续复性操作条件下可使进料质量浓度为1mg/mL的变性酶获得87%的总复性收率。     

Refolding of recombinant human interferon-γ by hydrophobic interaction chromatography

重组人γ-干扰素（rhIFN-γ）在大肠杆菌中高效表达,并形成包涵体.利用疏水作用层析（HIC）法对rhIFN-γ 进行复性.采用了等尿素梯度和线性尿素梯度两种复性方法,考察了线性尿素梯度下尿素梯度长度、尿素终浓度、流速和上样量对rhIFN-γ复性的影响.在优化的线性尿素梯度复性条件下,尿素浓度在10个柱体积内从6 mol•L^-1下降到2 mol•L^-1,流速为1 ml•min^-1、上样量为0.568 mg时,rhIFN-γ的活性收率比稀释复性法提高6.5倍,蛋白质量收率为36%,比活达1.9×10⁸ IU•mg^-1.     

疏水层析法复性重组人γ-干扰素

重组人γ-干扰素（rhIFN-γ）在大肠杆菌中高效表达,并形成包涵体.利用疏水作用层析（HIC）法对rhIFN-γ 进行复性.采用了等尿素梯度和线性尿素梯度两种复性方法,考察了线性尿素梯度下尿素梯度长度、尿素终浓度、流速和上样量对rhIFN-γ复性的影响.在优化的线性尿素梯度复性条件下,尿素浓度在10个柱体积内从6 mol•L^-1下降到2 mol•L^-1,流速为1 ml•min^-1、上样量为0.568 mg时,rhIFN-γ的活性收率比稀释复性法提高6.5倍,蛋白质量收率为36%,比活达1.9×10⁸ IU•mg^-1.     

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％。     

L-精氨酸助溶菌酶复性过程动力学研究

通过测定复性后酶活性和计算蛋白复性收率,研究了L-精氨酸助溶菌酶的最佳复性条件,利用蛋白质的复性和聚集反应竞争三态动力学模型描述了L-精氨酸助溶菌酶复性动力学,在溶菌酶浓度为100~500μg.mL-1、L-精氨酸浓度为0~1.0 mol.L-1的条件下,分析了溶菌酶浓度及L-精氨酸浓度对复性动力学常数的影响。结果表明,L-精氨酸助溶菌酶复性符合三级聚集反应动力学,L-精氨酸的主要作用是抑制蛋白聚集体的生成速率,从而达到抑制蛋白沉淀、提高复性收率的效果。     

疏水性电荷诱导层析纯化免疫球蛋白IgY

利用疏水性电荷诱导层析（HCIC）从鸡血中分离免疫球蛋白IgY。采用经辛酸预处理后的血浆上清液作为原料,直接用HCIC分离纯化IgY。考察了两种HCIC介质,市售介质MEP HyperCel和实验室自制介质Bestarose DVS MMI,优化了上样pH、洗脱pH和上样量等条件。研究发现,以2 巯基 1 甲基咪唑为配基的Bestarose DVS MMI介质优于MEP HyperCel介质,前者分离IgY的纯度和收率较高,HPLC分析IgY纯度可达92%以上,收率约95%。通过配基结构比较和表面电势分析,探讨了HCIC分离IgY的机制。结果表明,采用HCIC可以实现IgY的高效分离,为从血液中分离免疫球蛋白提供了新方法。     

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%。     

改进葡聚糖接枝技术制备高性能层析介质

利用改进的葡聚糖接枝技术,在以环氧氯丙烷为交联剂交联琼脂糖微球骨架的过程中加入葡聚糖溶液,在交联的同时接枝葡聚糖制得葡聚糖接枝型琼脂糖微球Rigose-Dex,再与盐酸2-氯三乙胺（DEAE）反应,获得葡聚糖接枝型高载量弱阴离子交换介质Rigose-Dex DEAE。以牛血清白蛋白（BSA）为模型蛋白,以商品化介质DEAE Sepharose 6FF为对照,系统研究了该葡聚糖接枝型Rigose-Dex DEAE的蛋白吸附性能,并进行了物理性能研究。结果表明,改进后葡聚糖接枝技术的最高接枝量为24.5mg/mL。自制Rigose-Dex DEAE可耐受700cm/h 的线性流速,对BSA的动态饱和载量为127.6mg/mL, 为商品介质DEAE Sepharose 6FF 载量的212%;具有在高流速下快速结合蛋白的能力,上样蛋白溶液在层析柱中停留2min即可基本达到饱和动态载量;重复使用性能好,经120 次在线清洗后,Rigose-Dex DEAE介质的动态载量为原始载量的90.4%。     

疏水性电荷诱导扩张床吸附分离免疫球蛋白IgY

结合疏水性电荷诱导色谱和扩张床吸附两项新型生物分离技术,开发了一个高效分离禽类血液免疫球蛋白IgY的新过程。以2-巯基-1-甲基咪唑（MMI）为疏水性电荷诱导配基,偶联于琼脂糖扩张床吸附基质上,制备出疏水性电荷诱导扩张床吸附剂S-MMI。结果表明,S-MMI具有较高的配基密度,约105 mmol·g^-1,pH7时IgY饱和吸附容量达71.26 mg·（ml介质）^-1,扩张床内可形成稳定的分级分布,床层稳定。选用经辛酸沉淀预处理后鸡血浆为料液,通过固定床色谱确定了上样和洗脱条件,比较了扩张床中不同膨胀率的影响,优化了分离条件,实现了扩张床吸附分离鸡血IgY,纯度达到98.9%,收率为80.5%。结果表明,疏水性电荷诱导色谱结合扩张床吸附,可以实现免疫球蛋白的高效分离,为禽类血液蛋白综合利用提供了新方法     

亲和层析介质上蛋白质偶联位点的控制方法

以溶菌酶为模型蛋白,将其偶联在环氧活化的琼脂糖介质上,采用液质联用技术结合蛋白质酶切技术对琼脂糖介质表面配基蛋白的偶联位点进行了识别,考察了活化时间、偶联时间和溶液pH值对偶联位点的影响. 结果表明,环氧基密度为11.34 mmol/g、反应pH 9.5条件下,溶菌酶偶联位点发生在K96,延长偶联反应时间蛋白配基偶联量增加,对偶联位点种类无显著影响;增加环氧基密度或提高偶联反应pH值使溶菌酶通过多种位点偶联,在pH≥10.5条件下偶联位点主要发生在K33, K96和K97.     

利用体积排阻色谱法进行蛋白质折叠

以溶菌酶为模拟体系对体积排阻色谱法进行蛋白质折叠过程实验研究 .圆二色性光谱法分析结果证实了复性溶菌酶与天然溶菌酶的二级结构一致性 ;复性溶菌酶与天然溶菌酶色谱保留体积的差异揭示出折叠过程中无活性蛋白质聚集体的存在及其向复性蛋白质转化的机制 ;不同初始浓度的复性实验证实了蛋白质聚集体的存在及其与变性蛋白质初始浓度的关系 ;采用短色谱柱的折叠分离实验结果表明蛋白质折叠是一个快速过程 ;不同尿素浓度下的折叠分离实验结果表明尿素在SEC法中具有非常重要的作用 .与稀释复性法的对比实验表明 :体积排阻色谱法具有稀释倍数小、复性产品活性收率高、复性蛋白质浓度高等优点 .     

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     

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     

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.     

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%.     

一种温敏性水凝胶复性体系:聚N-异丙基丙烯酰胺的制备及其在溶菌酶复性中的应用

Temperature-sensitive hydrogel-poly(N-isopropyl acrylamide) (PNIPA) was prepared and applied to protein refolding. PNIPA gel disks and gel particles were synthesized by the solution polymerization and inverse suspension polymerization respectively. The swelling kinetics of the gels was also studied. With these prepared PNIPA gels, the model protein lysozyme was renatured. Within 24 h, PNIPA gel disks improved the yield of lysozyme activity by 49.3% from 3375.2 U.mg-1 to 5038.8 U.mg-1. With the addition of faster response PNIPA gel beads,the total lysozyme activity recovery was about 68.98% in 3h, as compared with 42.03% by simple batch dilution.The novel refolding system with PNIPA enables efficient refolding especially at high protein concentrations. Discussion about the mechanism revealed that when PNIPA gels were added into the refolding buffer, the hydrophobic interactions between denatured proteins and polymer gels could prevent the aggregation of refolding intermediates,thus enhanced the protein renaturation.     

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.