title: Expression system for recombinant human growth hormone production from Bacillus subtilis.
abstract: 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.
authors.affiliation: Biochemical Reaction Engineering Laboratory, Chemical Engineering Dept., Ankara University, 06100 Ankara, Turkey. ozdamar@eng.ankara.edu.tr
authors.forename: Tunçer H
authors.initials: TH
authors.lastname: Ozdamar
authors.forename: Birgül
authors.initials: B
authors.lastname: Sentürk
authors.forename: Ozge Deniz
authors.initials: OD
authors.lastname: Yilmaz
authors.forename: Güzide
authors.initials: G
authors.lastname: Calik
authors.forename: Eda
authors.initials: E
authors.lastname: Celik
authors.forename: Pinar
authors.initials: P
authors.lastname: Calik
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics; D005285:Fermentation; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D010450:Endopeptidases; C087822:alkaline protease