EXPRESSION OF COMPLETELY GAMMA-CARBOXYLATED AND BETA-HYDROXYLATED RECOMBINANT HUMAN VITAMIN-K-DEPENDENT PROTEIN-S WITH FULL BIOLOGICAL-ACTIVITY

被引：23

作者：

MALM, J ^{[1
]}

COHEN, E ^{[1
]}

DACKOWSKI, W ^{[1
]}

DAHLBACK, B ^{[1
]}

WYDRO, R ^{[1
]}

机构：

[1] INTEGRATED GENET INC, FRAMINGHAM, MA USA

来源：

EUROPEAN JOURNAL OF BIOCHEMISTRY | 1990年 / 187卷 / 03期

关键词：

D O I：

10.1111/j.1432-1033.1990.tb15361.x

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Human anticoagulant vitamin‐K‐dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full‐length cDNA construct was introduced into the vector in the 5′ untranslated region of the mouse metallothionein‐I gene. Transfected cells expressed approximately 10 μg/ml of the recombinant protein which was purified by ion‐exchange chromatography followed by affinity chromatography using Ca2+‐dependent monoclonal antibodies against the region of protein S containing 4‐carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide‐gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N‐linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino‐terminal sequences, similar amino acid composition and number of 4‐carboxyglutamyl and 3‐hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b‐binding protein with the same apparent Kd. Protein S is the most extensively post‐translationally modified vitamin‐K‐dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity. Copyright © 1990, Wiley Blackwell. All rights reserved

引用

页码：737 / 743

页数：7

共 61 条

[51] BETA-HYDROXYASPARAGINE IN DOMAINS HOMOLOGOUS TO THE EPIDERMAL GROWTH-FACTOR PRECURSOR IN VITAMIN-K-DEPENDENT PROTEIN-S [J].