Taheri-Anganeh Mortaza, Khatami Seyyed Hossein, Jamali Zeinab, Savardashtaki Amir, Ghasemi Younes, Mostafavi-Pour Zohreh
Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
Recombinant Proteins Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biol Res Commun. 2019 Mar;8(1):17-26. doi: 10.22099/mbrc.2019.31801.1372.
An elevated cholesterol level might lead to cardiovascular disease (CVD). Statins block the cholesterol synthesis pathway in the liver. Atorvastatin is the most widespread statin worldwide and, its chemical synthesis requires toxic catalysts, resulting in environmental pollution. Hence, enzymatic synthesis of atorvastatin is desirable. This process could be done by alcohol dehydrogenase (LKADH). Therefore, recombinant enzyme secretion by using signal peptides (SPs) might result in easy production and purification. To achieve this objective, we used some online bioinformatics web servers to evaluate the suitable SPs for translocation of LKADH into extracellular spaces. "Signal Peptide Website" and "UniProt" were utilized to retrieve the SPs and LKADH sequences. "SignalP 4.1" was used to determine SPs and their cleavage site location and the results were rechecked by "Philius". Physicochemical features of SPs were evaluated by "ProtParam", then solubility of their fusion with LKADH was assessed by "Protein-sol". Finally, secretion pathway and sub-cellular localization of the selected stable and soluble LKADH fusions were predicted by "PRED-TAT" and "ProtCompB". Amongst the 41 evaluated SPs, only LPTA_ECOLI, SUBF_BACSU, CHIS_BACSU, SACB_BACAM, CDGT_BACST and AMY_BACLI could translocate LKADH out of cytoplasm. The six selected SPs in the result section were suitable to design a soluble secretory LKADH that accelerate its scale-up production and might be useful in future experimental researches.
胆固醇水平升高可能会导致心血管疾病（CVD）。他汀类药物可阻断肝脏中的胆固醇合成途径。阿托伐他汀是全球使用最广泛的他汀类药物，其化学合成需要使用有毒催化剂，会造成环境污染。因此，阿托伐他汀的酶促合成是可取的。这个过程可以通过乙醇脱氢酶（LKADH）来完成。因此，利用信号肽（SPs）进行重组酶分泌可能会使生产和纯化变得容易。为实现这一目标，我们使用了一些在线生物信息学网络服务器来评估适合将LKADH转运到细胞外空间的信号肽。利用“信号肽网站”和“UniProt”来检索信号肽和LKADH序列。使用“SignalP 4.1”来确定信号肽及其切割位点的位置，并通过“Philius”对结果进行重新检查。通过“ProtParam”评估信号肽的理化特性，然后通过“Protein-sol”评估其与LKADH融合后的溶解性。最后，通过“PRED-TAT”和“ProtCompB”预测所选稳定且可溶的LKADH融合蛋白的分泌途径和亚细胞定位。在评估的41种信号肽中，只有LPTA_ECOLI、SUBF_BACSU、CHIS_BACSU、SACB_BACAM、CDGT_BACST和AMY_BACLI能够将LKADH转运出细胞质。结果部分所选的六种信号肽适合设计一种可溶的分泌型LKADH，这将加速其扩大生产，并且可能在未来的实验研究中有用。
