【作者】 余路；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2007， 硕士

【摘要】 如何对富含二硫键的包涵体蛋白进行高效的体外复性是重组蛋白生产所面临的巨大挑战。DsbA是大肠杆菌周质胞腔中辅助多种含有二硫键的蛋白质正确折叠并具有生物学活性的一种二硫键异构酶。本文将DsbA质粒成功转化后，对转化子进行了发酵和纯化的优化，然后将制备的重组折叠酶DsbA初步应用于模型蛋白溶菌酶的体外复性之中。首先，将含编码dsbA基因的质粒pAVD63转化到E．coli BL21(DE3)，目标蛋白DsbA获得了可溶性表达。然后，利用统计实验设计的方法将生产重组DsbA的发酵过程进行了优化。通过Plackett-Burman设计挑选出了对DsbA表达量影响较大的4个因素，利用杂合设计进行实验，并通过拟合得到了响应曲面函数，但该函数的驻点是鞍点，因此不具有全局的极值。通过约束优化得到了较佳的实验点，在该实验点下DsbA的表达量比基本培养条件下提高了50.14％。再次，离心收集细胞后进行超声波破胞，将细胞破碎的清液经过离子交换层析得到纯化的DsbA。为提高DsbA分离纯化的效率、回收率并缩短DsbA的制备周期，对离子交换层析过程利用Box-Behnken设计进行了统计优化，考察了pH值、洗脱梯度、初始盐浓度和上样量的影响。以DsbA洗脱峰面积为响应值，拟合出响应面函数，并利用多目标规划中的功效系数法综合考虑以上3个目标，得到了较优的实验条件为：20mL Q Sepharose Fast Flow凝胶柱，pH值为8.3，洗脱高盐含量(B液所占比例)为5.88％，初始盐浓度为16.7mM，上样量为9.25mL。验证实验表明，得到的DsbA洗脱峰面积为4301.8mAU·mL，与模型预测的的相对误差仅为±2.4％。最终纯化后可得到377.9±1.74mg DsbA／L发酵液，蛋白收率为96.8％，纯度大于95％。通过肽质量指纹谱分析所得目标产物为高纯度的DsbA，分子量为23kD。最后，将DsbA应用于模型蛋白溶菌酶的复性过程中。利用Plakett-Burman设计考察了影响复性后溶菌酶蛋白回收和比活的9个因素，筛选出5个重要因素：初始溶菌酶浓度、尿素浓度、KCl浓度、GSSG浓度、GSH与GSSG摩尔比，并进一步对这5个重要因素进行了考察。在优化的复性条件下，当初始溶菌酶浓度为250μg／mL时，DsbA介导体系复性效果大大高于直接稀释复性的结果，溶菌酶活性回收率从44.52％提高到75.85％，为富含二硫键的包涵体蛋白体外复性提供了一种新的方法。更多还原

【Abstract】 How to refold inclusion body proteins with many pairs of disulfide bonds into natural conformation with high efficiency is a challengeable problem at present. DsbA, existing in the periplasm of E. coli, is a kind of disulfide bond isomerase, which assists the folding of the disulfide bond containing proteins to achieve biological activities. In the thesis, the plasmid containing the gene of dsbA was successfully transformed into E.coli, the culture conditions of the DsbA transformant in the fermentation and purification were optimized, then the purified DsbA was applied to assist the renaturation of lysozyme in vitro.Firstly, the plasmid pAVD63 coded with dsbA gene was tramsformed into E.coli BL21 (DE3) and the target protein was expressed intracellularly.Secondly, statistical experimental designs were used to optimize the fermentation process of recombinant DsbA in E.coli. Four most important parameters, which affect the DsbA expression, were screened among 17 parameters by Plackett-Burman design. Then hybrid design was applied to fit a response surface function, where stationary point was a saddle point with neither global maximum nor minimum. Finally, the optimal settings of fermentation parameters were calculated using the nonlinear constrained optimization method. Verification experiment showed that the settings resulted in 50.14% higher expression after the optimization.Thirdly, after the cells were collected by centrifugation and disrupted by ultrasonic with ice bath, DsbA was purified by ion exchange chromatograpy (IEC). In order to improve the efficiency and DsbA recovery of the purification process and shorten the production period, the IEC process was optimized by Box-Behnken design, the influence of pH, step of elution, initial salt concentration and loading amout were studied. The area of DsbA elution peak was set as the response to fit the response function, and three objects were integrated together by effectiveness coefficient method in MOP (Mutiple Objective Programming) and finally the optimized process was achieved: 20mL Q Sepharose Fast Flow gel packed column, pH 8.3, elution at step of 5.88% (percent of the buffer B, pH 8.3, 16.7mM Tris-HCl, 2M KCl), initial salt concentration 16.7mM, loading amount 9.25mL. The verification experiment showed the setting resulted in 4301.8 mAU·mL of the area of the DsbA elution peak, while the error was only ±2.4% compared with the predicted value. In the whole process, 377.9±1.74mg purified DsbA per liter fermentation broth was obtained, andthe protein recovery was 96.8%, the purity was more than 95%. The high purity of DsbA was identified by peptide mass finger print (PMF), and the molecular weight was ascertained to be 23kD.Finally, DsbA was applied to assist the refolding of the model protein lysozyme. Plackett-Burman design was employed to investigate 9 factors which influenced the lysozyme protein recovery and specific activity, and 5 most important factors were screened out: initial lysozyme concentration, urea concentration, KC1 concentration, GSSG concentration and the molar ratio of GSH to GSSG. Furthermore, these five significant factors were studied in details. At optimized experimental conditions, the activity recovery was increased from 44.52% to 75.85% in this DsbA mediated refolding system compared with direct dilution refolding when the initial lysozyme concentration was 250 μg/mL, This research provides an alternative method for the refolding of recombinant proteins expressed as inclusion bodies in vitro with many pairs of disulfide bonds.更多还原