Zhuo Xiao-Fa, Zhang Yi-Ying, Guan Yi-Xin, Yao Shan-Jing
J Biotechnol. 2014 Dec 20;192 Pt A:197-203. doi: 10.1016/j.jbiotec.2014.10.028.
Reteplase is the third generation of thrombolytic medicine and has many advantages over commercial t-PA. However, over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies due to nine pairs of disulfide bonds formation that is the main obstacle for correct folding. In this paper, in order to enhance soluble expression of recombinant reteplase in E. coli, DsbA/DsbC foldases were used to introduce disulfide bonds into the reduced polypeptide chain and catalyze their isomerization to the native disulfide linkage during the folding process. Firstly multiple E. coli protein expression systems, i.e. DsbA, DsbC and DsbA/DsbC co-expression were constructed. Subsequently, IPTG and l-arabinose were added to induce expression of foldases and reteplase accordingly, and experimental parameters such as culture temperature and inducer concentration were optimized. As a result, the co-expression system markedly enhanced soluble expression of recombinant reteplase, and up to 60% of reteplase achieved soluble expression especially for the DsbC co-expression system. The fibrin plate method for active reteplase quantification showed that ∼70 mg soluble reteplase per liter fermentation broth was obtained with 2.35 × 105 IU/mg thrombolytic activity. Finally, fluorescence spectra indicated that the structural conformation of soluble reteplase was identical to its native state. The soluble expression of recombinant reteplase in E. coli was accomplished by co-expression with DsbA/DsbC, which contributes to further research in clinical application and folding mechanism, and provides guidance for production of other proteins with disulfide bonds.
瑞替普酶是第三代溶栓药物，与市售的组织型纤溶酶原激活剂（t-PA）相比具有许多优势。然而，在大肠杆菌中过量表达重组瑞替普酶时，由于九对二硫键的形成，总是会以包涵体的形式积累，这是正确折叠的主要障碍。本文为了提高重组瑞替普酶在大肠杆菌中的可溶性表达，利用二硫键异构酶A（DsbA）/二硫键异构酶C（DsbC）在折叠过程中向还原的多肽链中引入二硫键并催化其异构化为天然二硫键连接。首先构建了多个大肠杆菌蛋白表达系统，即DsbA、DsbC以及DsbA/DsbC共表达系统。随后分别添加异丙基-β-D-硫代半乳糖苷（IPTG）和L-阿拉伯糖相应地诱导折叠酶和瑞替普酶的表达，并对培养温度和诱导剂浓度等实验参数进行了优化。结果表明，共表达系统显著提高了重组瑞替普酶的可溶性表达，尤其是DsbC共表达系统，高达60%的瑞替普酶实现了可溶性表达。用纤维蛋白平板法对活性瑞替普酶进行定量分析表明，每升发酵液可获得约70mg可溶性瑞替普酶，溶栓活性为2.35×105IU/mg。最后，荧光光谱表明可溶性瑞替普酶的结构构象与其天然状态相同。通过与DsbA/DsbC共表达实现了重组瑞替普酶在大肠杆菌中的可溶性表达，这有助于其在临床应用和折叠机制方面的进一步研究，并为其他含二硫键蛋白的生产提供指导。
