FASN基因的原核表达及生物信息学分析

Prokaryotic Expression and Bioinformatics Analysis of FASN Gene

脂肪酸合成酶(FASN)在生物体内起着重要的作用,主要参与恶性肿瘤的数量调控。本研究旨在构建pET28a-FASN原核表达载体,并表达重组His-FASN蛋白,对该基因进行结构与功能的生物信息学分析。设计FASN基因特异性引物,通过PCR扩增获得的目的基因与原核表达载体pET28a连接,经IPTG诱导表达His-FASN蛋白。获得基因片段大小为1 320 bp,编码440个氨基酸;成功构建至pET28a原核表达载体,通过优化表达,确定在温度为35℃、IPTG浓度0.5 mmol/L、诱导时间为6 h的条件下融合蛋白表达量较高,获得蛋白大小约为53 kD;生物信息学分析结果表明FASN基因编码的蛋白是一个不稳定且具有亲水性的蛋白,不存在信号肽及跨膜区,可成为蛋白激酶磷酸化位点有12个Ser、5个Thr、3个Tyr。此外,从蛋白相互作用网络中发现,相互作用的蛋白包括主要酰基辅酶A合成酶长链家族成员及乙酰辅酶A羧化酶家族成员,为开发抑制剂药物提供了理论依据。

Fatty acid synthetase(FASN) plays an important role in organisms, which is mainly involved in the quantitative regulation of malignant tumors. The aim of this study was to construct pET28 a-FASN prokaryotic expression vector, to express recombinant His-FASN protein, and to analyze the structure and function of the gene by bioinformatics. FASN gene specific primers were designed, and the target gene obtained by PCR amplification was connected with prokaryotic expression vector pET28 a and induced to express His-FASN protein by IPTG. The size of the gene fragment was 1 320 bp, and 440 amino acids were encoded. The pET28 a prokaryotic expression vector was successfully constructed. By optimizing expression, the expression of fusion protein was higher at 35℃,0.5 mmol/L IPTG and induction time 6 h, and the size of the protein was about 53 kD. The results of bioinformatics analysis showed that the protein encoded by the FASN gene was an unstable and hydrophilic protein. There is no signal peptide and transmembrane region. There are 12 Ser, 5 Thr and 3 Tyr, which can become phosphorylation sites of protein kinase. Moreover, the protein interaction network indicated that ineteracting proteins include members of the acyl coenzyme A synthetase long chain family and the member of the acyl coenzyme A carboxylase family, which laid the foundation for the development of inhibitor drugs.