Efficient secretion of the authentic mature human growth hormone by Bacillus subtilis

Abstract

We have constructed a secretion vector in Bacillus subtilis, designated pNPA225, which is based on Bacillus amyloliquefaciens neutral protease gene. In this vector, a unique StuI site is located at the 3′-terminal of the signal peptide-coding region, which can be used for accurate fusion of a heterologous gene to the signal sequence. Using this vector, the plasmid phGH526 containing a mature human growth hormone (hGH) gene was constructed. This plasmid could transform B. subtilis to secrete hGH efficiently into the medium. The size and N-terminal amino acid sequence of the secreted hGH were the same as those of the authentic one. We found that the larger hGH, considered to be a hybrid precursor, was present in the cell membrane as a major species. The secreted hGH was biologically active, possessing the same specific activity as the authentic hGH on the growth stimulation of a rat lymphoma cell. These results suggested that the signal peptide-hGH fusion could be translocated across the cell membrane and processed precisely in B. subtilis. The secretion level reached 40 mg l −1 in a high-density culture of B. subtilis transformed with phGH526.