Abstract
The capsid of SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) is composed of four structural proteins, including the spike glycoprotein (S protein), small envelope protein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein). These 4 structural proteins might be the antigenic determinants of SARS-CoV, thus they might be capable of inducing immune-responses against SARS-CoV. In this study, 4 recombinant baculoviruses Bac-SH, Bac-EH, Bac-MH and Bac-NH were constructed to express 4 Histidine-tagged S (rSH), E (rEH), M (rMH) and N (rNH) proteins, respectively, in insect cells. rEH, rMH and rNH expressed in Sf-9 cell were localized in the organelle, nucleus and plasma membrane, respectively. The maximum specific yield of rNH (90 μg/106 cells) was 7-fold higher than that of rEH (12.5 μg/106 cells), probably due to the variation in their localization. The differences in the intrinsic properties also resulted in the difference in the extraction process of rEH and rMH. rNH could be readily solublized by native binding buffer, and 2.75 mg rNH could be purified from 100 ml infected cells with a purifity of 90% by Immobilized Metal Affinity Chromatography (IMAC). However, rEH required the combination of a two-stage process and the use of urea for efficient extraction, and 0.66 mg rEH was purified from 200 ml infected cells with a purity of 72% by IMAC. In contrast, serious protein aggregation hindered the detection and purification of rMH. Purified rEH and rNH could be used in the development of SARS diagnostic reagents and possibly in the development of SARS vaccines.