【作者】 谭培生；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 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使其获得正确的空间结构是重组蛋白生产所面临的巨大挑战,现已成为制约生物工程技术产业化的瓶颈问题之一。本文以核糖核酸酶A（RNase A）为蛋白质体外重折叠的模型蛋白,对其体外重折叠过程进行了研究,探讨了这类目标蛋白复性过程的一些规律性的问题。 首先利用脲和DTT作为变性剂,研究了脲和DTT浓度对RNase A的酶活和自由巯基的影响规律。确定了初始蛋白浓度为10mg/mL的RNase A完全变性条件为8M脲,20mM DTT。 其次,利用完全变性的RNase A研究了体外稀释复性过程,在蛋白复性初始浓度为100μg/mL、pH8.0、GSH浓度为2mM、GSH/GSSG比例为4∶1～2∶1、脲浓度为0.5～2.0M时具有较好的复性效果,活性收率可达初始酶活的80～90%。利用荧光光谱法表征了复性过程蛋白质结构的变化,从荧光光谱和酶活变化规律分析可知:蛋白质复性过程首先是二级结构和疏水结构的形成,然后才是三级结构和二硫键的形成。利用二级聚集反应动力学模型对实验数据进行拟合,经计算得到浓度在0.1～2.0mg/mL范围的RNase A,拟合相关系数为0.98～0.998。随着复性液中蛋白浓度的增加,聚集沉淀速率加快,正确折叠速率减慢;此外,在复性液中添加低浓度的脲能有效促进RNase A的正确折叠。 再次,研究了折叠酶DsbA的制备,包括质粒转化、发酵培养、细胞收集、破胞和离子交换层析等,对发酵和纯化工艺进行了优化,获得了高纯度的目标蛋白DsbA,其浓度为176mg/L,分离过程收率为94.4%,纯度达到95%以上,DsbA产量达到了365±1.6mg/L发酵液。 最后,在复性缓冲液中添加DsbA辅助变性RNase A体外重折叠。实验结果表明DsbA不仅能提高多肽链折叠的速率,还能提高复性后酶活收率,这是由DsbA的氧化性、异构酶的性质,兼具分子伴侣的功能所决定的。由复性动力学模型对实验数据的拟合结果表明,DsbA的存在能提高正确折叠的速率常数k₂,而对形成聚集体的速率常数k₃影响较小,因此其综合效果是k₂/k₃的增大,促进了多肽链向正确的空间结构折叠,形成具有天然态结构的蛋白质分子。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, and it is becoming one of the industrial bottlenecks in bioengineering field. In this thesis, ribonuclease A (RNase A) was employed as a model protein to understand the formation of correct three-dimensional structure in the refolding of recombinant proteins in vitro. On this basis, the refolding mechanism in vitro was discussed.At first, RNase A was denatured by urea and dithiothreitol (DTT). Effect of strong denaturant urea and DTT on enzyme activity and free thiol number was investigated. When the initial concentration of RNase A was 10mg/mL, 8M urea and 20mM DTT can be used to make it denatured completely.Denatured RNase A was diluted in excess so that it can recover the natural conformation, 80-90% activity yield was achieved under the optimum condition, i.e. 100μg/mL denatured RNase A, pH 8.0, 2mM GSH, the ratio of GSH to GSSG from 4:1 to 2:1, 0.5-2M urea in the refolding by direct dilution. Also, the structure of RNase A along with the in vitro refolding was studied by spectrofluorimetry. Deducing from the changes of fluorescence spectrum and enzyme activity during the process, the mechanism of protein refolding can be speculated. At the beginning of renaturation, secondary structure and hydrophobic domain were formed immediately, after that tertiary structure and disulfide bonds formed so as to make active sites. From the experimental data of the renaturation of RNase A with the initial concentrations ranging from 0.1 to 2.0mg/mL, the fit coefficient of second-order aggregation model ranged 0.98-0.998. Along with the increase of protein concentration, aggregation rate also increased but the rate of correct folding decreased. The existence of low concentration of urea had stronger inhibition to aggregation than to correct refolding pathway, so that it can improve the activity recovery of renaturation.Then the fermentation and purification of recombinant DsbA protein were studied. The procedure included the transformation of plasmid, fermentation, collection of cells, disruption of cells and ion-exchange chromatography (IEC) separation. As a result of the optimization of the operation conditions of fermentation and separation, high purity of DsbA was obtained, with the final concentration of 176mg/L, the separation recovery 94.4%, the purity 95% and the production yield of 365.9±1.6mg/L fermentation broth.At last, DsbA-assisted refolding of RNase A was investigated in vitro. The results showed that DsbA not only improved the refolding rate of the peptides, but also improved the activity recovery of RNase A. The reason was that DsbA had extremely high oxidizing ability and could catalyze free cysteines to form disulfide bonds. Inaddition, DsbA also played the role of protein disulfide isomerase and chaperone so that mismatched disulfide bonds could be shuttled and aggregation rate would decrease. Fitting the experimental data with the kinetic model, the results showed that the addition of DsbA could increase the refolding kinetic constant k2, but had less effect on the aggregation kinetic constant k3, so that the ratio of k2/k3 was increased as DsbA concentration increased. As a result, DsbA helped to improve the activity recovery.更多还原