Expression and purification of SARS coronavirus proteins using SUMO-fusions.
Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.
Zuo
Xun
X
LifeSensors, Inc., Malvern, PA 19355, USA.
Mattern
Michael R
MR
Tan
Robin
R
Li
Shuisen
S
Hall
John
J
Sterner
David E
DE
Shoo
Joshua
J
Tran
Hiep
H
Lim
Peter
P
Sarafianos
Stefan G
SG
Kazi
Lubna
L
Navas-Martin
Sonia
S
Weiss
Susan R
SR
Butt
Tauseef R
TR
D000086782:Coronavirus 3C Proteases; D000086462:Coronavirus Nucleocapsid Proteins; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D019590:Nucleocapsid Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D000086462:Coronavirus Nucleocapsid Proteins; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C578557:spike glycoprotein, SARS-CoV; C033223:polyhistidine; D006639:Histidine; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases