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doi:10.1016/j.pep.2007.07.002    
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Copyright © 2007 Elsevier Inc. All rights reserved.

Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris

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Xueling Yua, b, 1, Zhaofa Lia, 1, Xiaobing Xiac, Hongqing Fanga, Corresponding Author Contact Information, E-mail The Corresponding Author, Changlin Zhoub and Huipeng Chena, Corresponding Author Contact Information, E-mail The Corresponding Author

aBeijing Institute of Biotechnology, 20 Dong Da Street, Fengtai District, Beijing 100071, China

bSchool of Life Science and Technology, China Pharmaceutical University, 24 Tong Jiaxiang, Nanjing 210009, China

cInstitute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom


Received 2 February 2007; 
revised 5 July 2007. 
Available online 16 July 2007.

Abstract

Ancrod is known as a thrombin-like enzyme from the venom of Calloselasma rhodostoma. The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPIC9 and was subsequently expressed in the yeast Pichia pastoris. Recombinant ancrod was produced in 5-L bioreactor using a sorbitol–methanol feeding strategy and recovered from the fermentation broth by hydrophobic, affinity, and ion exchange chromatography. SDS–PAGE analysis revealed that ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43–48 kDa which decreased to about 29 kDa after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein.

Keywords: Snake venom thrombin-like enzyme; Recombinant ancrod; Pichia pastoris

Article Outline

Materials and methods
Materials
Construction of ancrod expression plasmid
Construction and selection of ancrod expression strain
Expression of recombinant ancrod
Purification of recombinant ancrod
SDS–PAGE of recombinant ancrod and deglycosylated recombinant ancrod assay
Fibrinogenolytic assay
Fibrinogen clotting assay
Zymographic assay
Results and discussion
Construction of ancrod expression plasmid
Transformation and selection for positive clones
Pichia expression of ancrod
Purification of recombinant ancrod
Biological characterization of ancrod
Conclusion
Acknowledgements
References




Corresponding Author Contact InformationCorresponding authors. Fax: +86 10 63833521.
1 These two authors contributed equally to this work.

 
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