Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology.
The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Mozhdeh
M
Zamani
Aydin
A
Berenjian
Shiva
S
Hemmati
Navid
N
Nezafat
Mohammad Bagher
MB
Ghoshoon
Fatemeh
F
Dabbagh
Milad
M
Mohkam
Younes
Y
Ghasemi
D003001:Cloning, Molecular / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D003470:Culture Media; D011994:Recombinant Proteins; D019382:Human Growth Hormone