Construction of the plasmid-free strain for human growth hormone production

• Published in: Biochimie 2016-09, Vol.128-129, p.148-153
• Main Authors: Schulga, A.A., Mechev, P.V., Kirpichnikov, M.P., Skryabin, K.G., Deyev, S.M.
• Format: Article
• Language: English
• Subjects: Bacteriophage T7 - genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gene Expression Regulation; Human growth hormone; Human Growth Hormone - biosynthesis; Human Growth Hormone - genetics; Humans; Lac Operon - genetics; Lambda Red recombineering; Mass Spectrometry - methods; Plasmid-free; Plasmids - genetics; Promoter Regions, Genetic - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Somatotropin
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2016.08.007

The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L−1·OD550−1) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain. •Plasmid-free E. coli strain, overproducing hGH was made.•The plasmid-free strain demonstrated higher productivity and better growth characteristics than the plasmid-based strain.•The protein produced by the plasmid-free strain was purified and characterized to be hGH.