Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.
The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of (15)N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ≈600 mg/L when the yeast cells were culture in a fermenter. The purified (15)N-labeled r-RGD-hirudin retained full biological activity and was uniformly labeled. Heteronuclear NMR of the (15)N-labeled r-RGD-hirudin was performed for the first time, and all signals in the heteronuclear single quantum coherence (HSQC) spectrum were successfully assigned.
Wang
Jue
J
Center of Analysis and Measurement, Fudan University, Shanghai 200433, China.
Zhang
Yanling
Y
Li
Shiyun
S
Liu
Xingang
X
Yan
Xiaomin
X
Song
Houyan
H
Yu
Min
M
Dai
Linsen
L
Mo
Wei
W
D001709:Biotechnology / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010843:Pichia / Q000378:metabolism; D015854:Up-Regulation
D006629:Hirudins; D009587:Nitrogen Isotopes; C544959:RGD-hirudin