Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.
To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.
Chuck
Chi-Pang
CP
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong.
Wong
Chi-Hang
CH
Chow
Larry Ming-Cheung
LM
Fung
Kwok-Pui
KP
Waye
Mary Miu-Yee
MM
Tsui
Stephen Kwok-Wing
SK
D000085962:Angiotensin-Converting Enzyme 2; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D012995:Solubility; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D007703:Peptidyl-Dipeptidase A; D000085962:Angiotensin-Converting Enzyme 2