Synthesis of bioactive seminalplasmin by expression of a newly constructed fusion gene.
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.
Preuss
K D
KD
Max-Planck-Institut für Biophysikalische Chemie, Abt. Molekulare Biologie, Göttingen.
Krauhs
E
E
Scheit
K H
KH
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D008297:Male; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012661:Semen; D029607:Seminal Vesicle Secretory Proteins
D011506:Proteins; D011993:Recombinant Fusion Proteins; D029607:Seminal Vesicle Secretory Proteins