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High-density fermentation
and functional characterization of a recombinant echistatin
mutant
Tao Yang1, Bo
Niu1,2, Niuliang Cheng1, Jun Xie1
and Lijun Yang1*
1Department
of Biochemistry and Molecular Biology; Key Laboratory of
Cellular Physiology (Shanxi Medical University), Ministry of
Education, Taiyuan, Shanxi 030001, China.
2Capital
Institute of Paediatrics, Beijing 100020, China.
*Corresponding author. E-mail:
biochemyy@126.com.
Tel: 86-351-4135452.
Fax:
86-351-4135452.
Accepted 12 May, 2009 |
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A
disintegrin echistatin mutant,
R24K-echistatin, was obtained by overlap extension polymerase
chain reaction. It was efficiently expressed as a soluble
and fusion protein in Escherichia coli at optimized
fermentation conditions. The recombinant bacteria had the
highest wet weight of 120 g/l in 2´YT
medium with IPTG induction. The R24K-echistatin was purified
by chitin
affinity chromatography and had a
yield of 13
mg/l
with DTT-cleavage. The purified R24K-echistatin inhibited
the aggregation of platelet in vitro
with IC50 of 21 nM which is better
efficient than echistatin.
This work lays the foundation for further research on
specific anti-thrombus disintegrin agents.
Key
words:
Disintegrin, echistatin,
Escherichia coli, fermentation, mutant. |
