High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells.
OBJECTIVE
To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.


METHODS
The human adenovirus vector was used to express the soluble S-protein (corresponding to 1 approximately 1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.


RESULTS
Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 microg/10(6) cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.


CONCLUSIONS
The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.
Department of Basic Veterinary Medicine, School of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, Hebei Province, China. feizhong2000@yahoo.com
Fei
F
Zhong
Zhen-Yu
ZY
Zhong
Shuang
S
Liang
Xiu-Jin
XJ
Li
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis; D045473:Severe acute respiratory syndrome-related coronavirus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
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