Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture

• High-level production of ggGH was performed by fed-batch culture.
• Purification of ggGH from inclusion bodies was performed by an easy method.
• Biologically active ggGH was obtained through a simple refolding process.
• Bioactive ggGH has growth-stimulating activity on human and teleost cells.

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30 h of cultivation, a cell concentration of 41.1 g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0 g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3 M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7 mg of ggGH was obtained from 10 ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC–MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.