Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon-alpha2b fusion protein.
In an attempt to increase the secretion capacity of Pichia pastoris (Pp), PpPMR1 gene was disrupted with GS115 as parent strain, and the resultant mutant was designated as Pppmr1. Pppmr1 displayed a Ca2+-dependent growth defect, which was consistent with the PMR1 mutation in other yeasts. HSA-L5-IFNalpha2b, a human serum albumin (HSA) and inferferon-alpha2b (IFNalpha2b) fusion protein with a flexible linker of 5 amino acid residues, was employed as a reporter to study the effects of PpPMR1 disruption on the secretion of heterologous protein. Because of its decreased viability after induction, Pppmr1 secreted more HSA-L5-IFNalpha2b only during the early phase (the first 15 hours) of induction. Although HSA-L5-IFNalpha2b secreted from GS115 and Pppmr1 had similar antiviral activity, the latter was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to aggregation at neutral to mild alkaline pH). Site-directed mutagenesis revealed that the heterogeneity of HSA-L5-IFNalpha2b secreted from Pppmr1 was originated from the incomplete disulphide bridge pairing between Cys1 and Cys98 of IFNalpha2b. To be secreted homogeneously from Pppmr1 and to be stable in aqueous solution, the linker of the fusion protein should be extended to 10 amino acid residues.
Zhao
Hong Liang
HL
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.
Xue
Chong
C
Wang
Yang
Y
Duan
Qing Fen
QF
Xiong
Xiang Hua
XH
Yao
Xue Qin
XQ
Liu
Zhi Min
ZM
D000998:Antiviral Agents / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D012709:Serum Albumin / Q000235:genetics* / Q000378:metabolism; D018116:Vesiculovirus / Q000502:physiology
D000998:Antiviral Agents; D005656:Fungal Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D000252:Calcium-Transporting ATPases