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门冬胰岛素原包涵体氧化复性的研究
陈颖
学位类型硕士
导师苏志国 ; 刘永东
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词门冬胰岛素 包涵体复性 二硒基化合物 复性添加剂 连续流复性
摘要

自胰岛素被发现以来,在近一个世纪的时间里,胰岛素及胰岛素类似物一直是治疗糖尿病不可缺少的药物。重组表达的胰岛素及其类似物已经占据了胰岛素市场的90% 以上。重组胰岛素可以通过原核体系和真核体系表达。原核体系有较高的生产速率,成本低廉,但是仅表达为无活性的包涵体。因此如何提高包涵体的复性收率,是大肠杆菌作为表达载体时的关键限制因素。本文以门冬胰岛素原作为研究对象,结合多种检测手段,分析其复性路径,并对不同复性条件对复性收率的影响进行了研究。通过对门冬胰岛素原氧化复性过程的研究,确定了两种二硫键错误配对导致的副产物,即由于分子间二硫键连接而形成的寡聚体以及分子内二硫键错配形成的二硫键异构单体。精氨酸以及低浓度的尿素与盐酸胍均被用于重组蛋白的复性中,但它们对复性的影响和作用机制有所不同。三种小分子都对门冬胰岛素原的溶解有促进作用,并抑制大分子聚集体的形成。但是与精氨酸相比,当添加剂为尿素时,复性过程中所产生的寡聚体尺寸更大。当复性缓冲液中氧化还原剂为5 mM GSSG与1 mM GSH,蛋白浓度为0.5 mg/mL时,尿素浓度由2 M提高至4 M,聚集体大量减少,但是复性收率反而由40%降低至30%。这主要是因为随着尿素浓度增加,另一种复性副产物,即二硫键异构体大量增加。盐酸胍对复性的影响与尿素相似。与之相对的,当精氨酸浓度达到1 M时,伴随着寡聚体量的减少,最终的复性收率提高至50%。另一方面,由于门冬胰岛素原中含有3对二硫键,需要在复性过程中提供适宜的氧化还原条件。在研究中发现,只在复性缓冲液中添加氧化剂即可完成复性过程,得到较高的复性收率。在0.5 M 精氨酸、蛋白浓度为0.5 mg/mL的情况下,添加10 mM GSSG以及5 mM 胱氨酸,可以使复性收率达到55%和60%;而使用5 μM硒代胱胺或硒代胱氨酸,均可使复性收率提高到80%。进一步研究发现,当使用2 M尿素代替0.5 M精氨酸时,也可以达到相同的复性收率,但是需要将蛋白浓度降低至0.3 mg/mL。复性后的门冬胰岛素原通过一步阴离子交换层析,纯度可达到95%,收率为32%。此外,为了简化操作步骤,本文发展了一种连续流复性纯化的方法。将层析系统的混合池作为复性反应器,使变性蛋白与复性缓冲液在此混合并在柱前管路中完成复性。通过调节管路的长度和流速可以控制目标蛋白的复性时间。使用这种连续复性纯化方法,可以得到与分步复性纯化相近收率和纯度的门冬胰岛素原。利用胰蛋白酶和羧肽酶B对纯化后的门冬胰岛素原进行酶切,并采用阳离子交换介质去除前导肽和C肽,获得纯度大于95% 的门冬胰岛素,质谱证实所得门冬胰岛素分子量与理值完全一致。

其他摘要

Since 1920s, insulin was found and used for diabetic patients. After nearly 100 years, insulin and its analogues are still the first choice for this disease. They are now mainly produced through genetic engineering techniques. Compared with yeast, the E.coli host system allows advantages such as high yield, low cost, simple media and easy to handle, but it always expresses proinsulin as inactive inclusion bodies. Hence, the refolding of proinsulin inclusion bodies has been recognized as the key unit for insulin's production. In this study, by characterizing the components during the refolding of proinsulin aspart, the precursor of a rapid-acting insulin analogue of insulin aspart, we clearly identified the disulfide-linked aggregates and disulfide-isomerized monomers (monomers with incomplete or incorrect disulfide bonds) as the only two off-path folding components.Arginine and low concentration of urea as well as guanidine hydrochloride are widely used additives presenting obvious advantage of inhibiting precipitation during protein refolding. They may have different effects on the protein refolding. In this work, at the protein concentration of 0.5 mg/mL, when the urea is above 2 M, guanidine hydrochloride above 0.5 M or arginine above 0.2 M, precipitation could be completely inhibited, resulting in almost 100% mass recovery. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased.For proinsulin aspart containing three disulfide bonds, redox system is another important factor on refolding yield. In this work, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin aspart. The maximum refolding yield of 55% or 60% was obtained only with 10 mM GSSG or 5 mM cysteine in the presence of 0.5 M arginine and at the protein concentration of 0.5 mg/mL。An oxidative agent of selenocystamine could increase the yield up to 80%. Considering arginine needs to be removed for the following ion exchange chromatographic purification,refolding with 2 M urea placing 0.5 M arginine was also investigated. When protein concentration is reduced to 0.3 mg/mL, similar yield could be achieved as in the presence of 0.5 M arginine.Refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. Moreover, a continuous refolding and purification process was established using the mixer of the purifier equipment as a tiny refolding vessel. By adjusting the tube length and sample loading speed, the recovery and purity for proinsulin achieved through this continuous process was similar to those through two units operated separately. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22866
专题研究所(批量导入)
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陈颖. 门冬胰岛素原包涵体氧化复性的研究[D]. 北京. 中国科学院研究生院,2016.
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