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Expression, purification
and characterization of recombinant targeting bifunctional
hirudin in Pichia pastoris
Zongli Hu, Ning Zhang,
Feng Gu, Yong Li, Xiaojie Deng and Guoping Chen*
Key
Laboratory of Biorheological Science and Technology
(Chongqing University), Ministry of Education,
Bioengineering College, Chongqing University, Chongqing
400044, People’s Republic
of China.
*Corresponding author. E-mail:
chenguoping@cqu.edu.cn. Tel: 0086 23 65112674. Fax: 0086
23 65102507.
Accepted
10 July, 2009 |
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A
recombinant targeting bifunctional hirudin was expressed in
the yeast Pichia pastoris. In order to decrease the
side effects of hirudin and increase its activity to prevent
arterial thrombus, we fused a factor Xa (FXa) recognition
sequence into N’ of hirudin, while maintaining the activity
of natural hirudin. In addition, an Arg-Gly-Asp (RGD)
sequence was fused into appropriate genetic locus of hirudin.
Furthermore, we added a 9 × His - Tag to make its separation
and purification conveniently. The recombination hirudin
gene was successfully cloned and ligated into the P.
pastoris vector pPIC9K to form an expression vector,
which was transferred into P. pastoris GS115. A
transformant strain was selected and expressed efficiently
in suitable conditions. Then, the fusion protein was
purified by affinity chromatography. Through anti-thrombin
activity analysis and anti-platelet aggregation activity
analysis, we found that the anti-thrombin activity of the
fusion protein did not change comparing with the natural
hirudin, whereas its anti-platelet capability was enhanced.
Key
words:
Targeting, bifunctional, hirudin, recombinant,
anticoagulant, Pichia pastoris. |
