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Abstract

A synthetic DNA, carrying the coding sequence for seminalplasmin (SAP), the major basic protein of bull semen, was cloned into the C-terminal part of a shortened, mutated fragment of the lacZ gene (lacZ-MF) of vector pLZPWB1. As a result of the mutation, all methionine as well as cysteine residues are replaced by other amino-acid residues. In the fusion gene lacZ-MF-SAP of the resulting construct pSAP4 the two proteins are linked through a methionine residue. Expression of pSAP4 in E. coli W3110 in the presence of the inducer isopropylthiogalactoside (IPTG) led to production of fusion protein with a yield of approximately 50% of the total proteins synthesized. All SAP-immunoreactive fusion protein was found within the insoluble protein fraction and represented 40% of total proteins produced during expression. The fusion protein was subjected to cyanogen bromide cleavage. The overall yield of crude SAP with a purity of 80% was 10 mg/l of culture. The crude SAP was further purified by calmodulin-Sepharose affinity absorption. Characterisation by protein chemical analysis indicated the identity of recombinant SAP with authentic SAP purified from bull semen.