Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis

The gene encoding subtilisin Carlsberg from Bacilluslicheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5′ and 3′ flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is proceeded by two putitative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.