We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.
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