Expression and purification of SARS coronavirus proteins using SUMO-fusions

Abstract

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.

Fig. 1

The procedure for purification of SARS-CoV proteins expressed with SUMO-fusion system in E. coli and cleavage of 6× His-SUMO-tagged proteins.

Fig. 2

Enhanced expression of SARS-CoV 3CL protease (3CL) by SUMO-fusion in E. coli. Cells grown in either Luria–Bertani (LB) or M9 minimal (MM) medium were induced at the temperatures and for the lengths of time indicated. Just before expression was induced and after induction was completed the cells from a 1.5 ml aliquot of culture were lysed. Samples of whole cell lysates (∼7.5 μl) from the various expression conditions were resolved in 12% SDS-gels and stained with Coomassie blue. Molecular weights were as indicated, and arrowheads highlight expected/observed positions of respective expressed protein bands.

Fig. 3

Enhanced expression of SARS-CoV Nucleocapsid protein (Nc) by SUMO-fusion in E. coli. The conditions for cell culture, protein expression, and gel detection were the same as in Fig. 2. The yields of expressed SUMO-Nc proteins were higher than the Nc expressed without SUMO in minimal media, but there were no significant differences in their expression in LB media.

Fig. 4

Enhanced expression of SARS-CoV Spike C protein (Spk C) by SUMO-fusion in E. coli. The cells were cultured in LB media and the conditions for cell culture, protein expression, and gel detection were the same as in Fig. 2. The Western blot (right panel) was performed with anti-His-tag primary antibody.

Fig. 5

Detection of proteins in samples from various steps of a typical purification of SARS-CoV 3CL protease. Aliquots of the samples (each containing ∼5 μg protein) were separated on a 12% SDS-gel and stained with Coomassie blue. The migration positions of the SUMO-fusion and the proteins resulting from the cleavage are as indicated.

Fig. 6

Detection of proteins from various steps of a typical purification of SAR-CoV Nucleocapsid proteins. Aliquots (∼5 μg proteins) of the samples were resolved in a 12% SDS-gel and stained with Coomassie blue. The migration positions of molecular weight markers, SUMO-fusion, and proteins resulting from the cleavage are as indicated.

Fig. 7

Detection of reactions of the purified Nc and 3CL proteins with the respective SUMO-fusion antibodies. The purified SARS-CoV proteins were detected on the Coomassie blue stained 12% SDS-gel (A) and the Western blots were probed with the SUMO-3CL antibody (B) and with the SUMO-Nc antibody (C). The amounts of the purified proteins loaded for the SDS-gel and the Western blots were 4 and 2  μg, respectively. Arrowheads highlight observed positions of respective SARS protein bands.

Fig. 8

Effect of different amounts of SUMO protease on cleavage of SUMO-Nc-fusion. Serially diluted SUMO protease was added to 10 μg of purified SUMO-Nc-fusion protein and incubated in 30 μl PBS containing 5 mM β-mercaptoethanol at 30 °C for 1 h. The protease used was 20 U/μg. Aliquots (∼12 μl) from the incubation mixture were resolved on a 12% SDS–polyacrylamide gel and stained with Coomassie blue. Lanes: 0, uncleaved fusion (control); 1, 2 U of the protease; 2, 1 U; 3, 0.5 U; 4, 0.25 U; 5, 0.125 U; 6, 0.063 U; 7, 0.032 U; 8, 0.016 U; 9, 0.008 U; 10, 0.004 U.

Fig. 9

Detection of SUMO-SARS CoV Spike C proteins from expressed E. Coli cells (A) and various steps during a representative purification of Spike C proteins (B). Aliquots (∼ 5  μg protein) of samples were resolved in 12% SDS-gels and stained with Coomassie blue. Lanes in (A): 1, uninduced cells (control); 2, induced cells; 3, insoluble fraction (the starting material for purification). Lanes in (B): M, molecular weight markers; 1, purified SUMO-Spike C-fusion; 2, cleaved SUMO-Spike C-fusion; 3, purified Spike C sample. The migration positions of the respective proteins are as indicated.