Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.
We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.
Chen
Chiu-Yueh
CY
Department of Biochemistry and Institute of Basic Medical Science, National Cheng Kung University College of Medicine, Tainan 701, Taiwan.
Cheng
Chun-Ho
CH
Chen
Yi-Chun
YC
Lee
Jenq-Chang
JC
Chou
Shan-Ho
SH
Huang
Wenya
W
Chuang
Woei-Jer
WJ
D003545:Cysteine / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics*; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010976:Platelet Count
D005656:Fungal Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; C072026:rhodostomin; D003545:Cysteine