High-level secretory expression, purification and characterization of Ailuropoda melanoleuca growth hormone in Pichia pastoris.
Growth hormone is one of the most important hormones, which is involved in many reproductive processes of giant panda Ailuropoda melanoleuca. In this study, the mature peptide of A. melanoleuca growth hormone (AmGH) was successfully expressed and secreted in Pichia pastoris under the control of AOX1 promoter. The expression condition for AmGH in P. pastoris, such as the expression time, pH value and methanol concentration in the BMMY were optimized and the AmGH expression level is about 100 mg/L using GS115 recombinant under optimized condition (96 h of 1.5% methanol induction). The secreted nascent AmGH were purified using ammonium sulfate fractionation. The mature AmGH protein exhibited a molecular mass of approximately 22 kDa on SDS-PAGE. This study would provide a new opportunity for large-scale expression and purification of AmGH, which might facilitate studies on the biological activity of AmGH.
Xu
Qinggang
Q
College of Life Sciences, Zijingang Campus, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, PR China.
Wu
Xuechang
X
Hou
Rong
R
Liao
Mingjuan
M
Il
Kim Hyok
KH
Shou
Jianxin
J
Bian
Hongwu
H
Han
Ning
N
Pan
Jianwei
J
Zhang
Zhihe
Z
Zhu
Muyuan
M
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001503:Ursidae
D017931:DNA Primers; D011994:Recombinant Proteins; D013006:Growth Hormone