Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris.
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.
Yu
Xueling
X
Beijing Institute of Biotechnology, 20 Dong Da Street, Fengtai District, Beijing 100071, China.
Li
Zhaofa
Z
Xia
Xiaobing
X
Fang
Hongqing
H
Zhou
Changlin
C
Chen
Huipeng
H
D000721:Ancrod / Q000235:genetics / Q000302:isolation & purification*; D000925:Anticoagulants / Q000302:isolation & purification*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D000925:Anticoagulants; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000721:Ancrod