Expression, purification and characterization of aprotinin and a human analogue of aprotinin.
Aprotinin is a Kunitz-type inhibitor with a relatively broad specificity. It has been shown to be clinically useful for the management of hemorrhagic complications. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant aprotinin and a human aprotinin analogue (cloned form human cDNA library). Both fusion proteins were overexpressed mainly as inclusion bodies in Escherichia coli and accounted for approximately 28% of the total cell proteins. After purification by Ni-Sepharose affinity chromatography and renaturation, the fusion proteins were cleaved with SUMO protease 1. Aprotinin and its analogue were separated from the fusion partner by the subtractive chromatography using Ni-Sepharose and then further purified with CM-cellulose. Kinetic studies demonstrated that the amidolytic activity of plasmin was competitively inhibited by recombinant aprotinin with a K(i) of 8.6+/-2.4 nM, which was similar to the K(i) (7.5+/-2.7 nM) of natural aprotinin. The K(i) of human aprotinin analogue was 22.7+/-6.5 nM. The expression strategy described in this study allows convenient high yield and easy purification of small recombinant protease inhibitors with complete native sequences.
Sun
Ziyong
Z
Institute of Molecular and Experimental Therapeutics, East China Normal University, 3663 Zhongshan North Road, Shanghai 200062, China.
Lu
Wei
W
Jiang
Aiqing
A
Chen
Junyong
J
Tang
Fengyuan
F
Liu
Jian-Ning
JN
D007611:Aprotinin / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005341:Fibrinolysin / Q000037:antagonists & inhibitors*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D025842:SUMO-1 Protein / Q000378:metabolism; D012995:Solubility
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D007611:Aprotinin; D005341:Fibrinolysin