Copyright © 2007 Elsevier Inc. All rights reserved.
Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris
Received 2 February 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
- 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






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