Engineering of Harobin for enhanced fibrinolytic activity obtained by   random and site-directed mutagenesis

doi:10.1016/j.pep.2015.09.010

CORC > 北京大学 > 生命科学学院

	Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis
	Li, Zhuojian ; Chen, Xiaojia ; Guo, Shujun ; Zhang, Huihua ; Dong, Haojun ; Wu, Guoyi ; Hong, An ; Gu, Jun
刊名	PROTEIN EXPRESSION AND PURIFICATION
	2017
关键词	Random mutagenesis Double mutant Harobin Thrombosis Hypertension ERROR-PRONE PCR SNAKE-VENOM HYPERTENSIVE-RAT PLATELET-AGGREGATION ORAL ANTICOAGULANTS PICHIA-PASTORIS EVOLUTION HEMOSTASIS PROTEINS IDENTIFICATION
DOI	10.1016/j.pep.2015.09.010
英文摘要	We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 degrees C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension. (C) 2015 Elsevier Inc. All rights reserved.; Ministry of Science and Technology of Guangdong Province [2009B090300284]; SCI(E); PubMed; ARTICLE; 162-172; 129
语种	英语
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/418113]
专题	生命科学学院
推荐引用方式 GB/T 7714	Li, Zhuojian,Chen, Xiaojia,Guo, Shujun,et al. Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis[J]. PROTEIN EXPRESSION AND PURIFICATION,2017.
APA	Li, Zhuojian.,Chen, Xiaojia.,Guo, Shujun.,Zhang, Huihua.,Dong, Haojun.,...&Gu, Jun.(2017).Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis.PROTEIN EXPRESSION AND PURIFICATION.
MLA	Li, Zhuojian,et al."Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis".PROTEIN EXPRESSION AND PURIFICATION (2017).