含有对叠氮苯丙氨酸的人内皮抑素的表达及纯化

优化定点引入对叠氮苯丙氨酸的詹氏甲烷球菌属正交酪氨酰tRNA合成酶/tRNA系统,使用优化后的正交系统在大肠杆菌内表达并纯化在特定氨基酸位点引入对叠氮苯丙氨酸的人内皮抑素突变体。首先将正交酪氨酰tRNA合成酶启动子-15区的TATAA序列突变为TTAA序列,同时将正交酪氨酰tRNA合成酶的第286位天冬氨酸突变为精氨酸以提高对抑制型tRNA反密码子CUA的识别力。然后将质粒pST和pET32a-mhEn转化到大肠杆菌DH10BDE3中得到重组工程菌。该重组菌在含有对叠氮苯丙氨酸的培养基中培养,并经IPTG诱导表达人内皮抑素突变体。利用镍亲和色谱纯化获得人内皮抑素突变体,纯度大于90%。通过对正交酪氨酰tRNA合成酶/tRNA系统的优化,有效提高了系统引入对叠氮苯丙氨酸的效率,人内皮抑素突变体的表达量有了显著提高。

Expression and purification of the human endostatin containing the p-azido-phenylalanine

To optimize the orthogonal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA system for site-specific incorporation of p-azido-phenylalanine (pAzPhe) in E. coli.The human endostatin mutant (mhEn) with pAzPhe inserted at specific amino acid site was expressed using the optimized system.The promoter of the orthogonal MjTyrRS was mutant to TTAA from TATAA at the -15 position.The Asp at 286 amino acid site of the orthogonal MjTyrRS was mutated to Arg to increase the recognition of the anticodon (CUA) in the amber suppressor tRNA of MjTyrRS.The recombinant plasmids pST and pET32a-mhEn were co-transformed into DH10BDE3.The mhEn was expressed in the medium containing pAzPhe after IPTG induction and the mhEn was purified by Ni-affinity chromatography with a purity of 90%.The optimized orthogonal MjTyrRS/tRNA system could incorporate pAzPhe with higher efficiency and the yield of mhEn was improved significantly.