Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast.
Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).
Yao
Xue Qin
XQ
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.
Zhao
Hong Liang
HL
Xue
Chong
C
Zhang
Wei
W
Xiong
Xiang Hua
XH
Wang
Zhi Wei
ZW
Li
Xian Yu
XY
Liu
Zhi Min
ZM
D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation; D020934:Ciliary Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020934:Ciliary Neurotrophic Factor; D005656:Fungal Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D016282:Aspartic Acid Endopeptidases