[Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fusion with Hela cell membrane].
OBJECTIVE
To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.


METHODS
S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.


RESULTS
The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.


CONCLUSIONS
The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.
Liu
Yun
Y
Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province, Southern Medical University, Guangzhou, China. sysliuyun@126.com
Liu
Ai-Hua
AH
Deng
Peng
P
Wu
Xiang-Ling
XL
Li
Tao
T
Liu
Ya-Wei
YW
Xu
Jia
J
Jiang
Yong
Y
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D050576:Membrane Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
D050576:Membrane Fusion Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D049452:Green Fluorescent Proteins