The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni(2+)-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166-180, 356-375, and 396-410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156-175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156-175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1-30, 86-100, 306-320, and 351-365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2K(d) CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-gamma secretion cell assay and analysis by flow cytometry, peptides containing residues 81-95 were found to be capable of stimulating both CD4(+) and CD8(+) cell proliferation in vitro. We also only observed that peptides corresponding to residues 336-350 were capable of stimulating IFN-gamma production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 59-end and XhoI at the 39-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His 6 -Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His 6 )-tagged Smt3. Smt3 is the yeast SUMO protein. His 6 -Smt3-X was purified by Ni 2+ resin. Removal of His 6 -Smt3 was performed on the Ni 2+ resin by an engineered SUMO protease, His 6 -Ulp1 403-621 -His 6 . Because of its dual His 6 tags, His 6 -Ulp1 403-621 -His 6 exhibits a high affinity for Ni 2 resin and associates with Ni 2+ resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni 2+ -resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.Keywords: fusion protein; SUMO; Rad51; RecA; enterovirus; foot-and-mouth disease virus In humans, many diseases, including cancer, aging, anemia, and developmental disorders arise because of gene mutations that result in aberrant proteins. Therefore, proteins are targets for therapeutic drugs, and protein production for structural and functional analysis is a major task in modern biology and medicine. Protein production is never a simple task because proteins are extremely diverse in their physio-chemical properties, and there is no generic 6 These authors contributed equally to this work. Reprint requests to: Ting-Fang Wang, Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; e-mail: tfwang@gate.sinica.edu.tw; fax: 886-2-27889759; or Chih-Hsiang Leng, Vaccine Research and Development Center, National Health Research Center, Miaoli 350, Taiwan; e-mail: leoleng@nhri.org.tw; fax: 886-37-583009.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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