Fazeli Behnaz, Akbari Vajihe, Barkhordari Asgar, Mir Mohammad Sadeghi Hamid
Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Adv Biomed Res. 2019 Oct 31;8:65. doi: 10.4103/abr.abr_212_18. eCollection 2019.
Reteplase is a nonglycosylated derivative of recombinant tissue plasminogen activator, a thrombolytic agent, which can be easily expressed in . However, overexpression of reteplase in usually leads to accumulation of insoluble and inactive aggregates and inclusion bodies. In the present study, we aimed to optimize chemical additives of lysis buffer to avoid the initial aggregation and formation of inclusion bodies of reteplase at cell disruption step.
After protein expression in BL21 (DE3), the bacterial cells were disrupted in different lysis buffers using microsmashing. Eleven chemical additives at two concentration levels were combined based on a Plackett-Burman design to prepare 12 different lysis buffers used at cell disruption stage. Then, three additives with the most positive effect on improvement of solubility of reteplase were chosen and used for the second screening based on Box-Behnken model.
The primary screening results showed that among 11 additives, arginine, KPO and cetyltrimethylammonium bromide (CTAB) had the most positive effect on solubility of reteplase. Our final results based on 14 runs of Box-Behnken design showed that the optimum buffer additive condition is 0.005 mg/ml CTAB, 0.065 mg/ml arginine, and 0.026 mg/ml KPO. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting of soluble and total fraction of samples confirmed that these additives significantly improved soluble production of reteplase compared with control.
Our study indicates that the application of chemical additives in cell lysis can improve the solubility of reteplase. Further studies are still required to understand the exact mechanism of chemical additives as a chemical chaperone during cell lysis.
瑞替普酶是重组组织型纤溶酶原激活剂的非糖基化衍生物，一种溶栓剂，它能够在……中轻易表达。然而，瑞替普酶在……中的过表达通常会导致不溶性和无活性聚集体及包涵体的积累。在本研究中，我们旨在优化裂解缓冲液的化学添加剂，以避免在细胞破碎步骤中瑞替普酶出现初始聚集和包涵体形成。
在BL21(DE3)中进行蛋白质表达后，使用微粉碎在不同的裂解缓冲液中破碎细菌细胞。基于Plackett-Burman设计将11种化学添加剂在两个浓度水平上进行组合，以制备12种用于细胞破碎阶段的不同裂解缓冲液。然后，根据Box-Behnken模型选择对瑞替普酶溶解度改善最具积极作用的三种添加剂用于二次筛选。
初步筛选结果表明，在11种添加剂中，精氨酸、磷酸钾和十六烷基三甲基溴化铵（CTAB）对瑞替普酶的溶解度具有最积极的影响。基于14次Box-Behnken设计的最终结果表明，最佳缓冲液添加剂条件为0.005 mg/ml CTAB、0.065 mg/ml精氨酸和0.026 mg/ml磷酸钾。对样品的可溶性和总组分进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析和蛋白质印迹法证实，与对照相比，这些添加剂显著提高了瑞替普酶的可溶性产量。
我们的研究表明，化学添加剂在细胞裂解中的应用可以提高瑞替普酶的溶解度。仍需要进一步研究以了解化学添加剂在细胞裂解过程中作为化学伴侣的确切机制。
