We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre-) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg-promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t = 32 h as C(rhGH) = 70 mg L(-1) with a product yield on substrate Y(rhGH/S) = 9 g kg(-1), in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species.
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