title: Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.
abstract: 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 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/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 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml 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.
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Wen-Jen
authors.initials: WJ
authors.lastname: Chung
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Chi-Lung
authors.initials: CL
authors.lastname: Huang
authors.affiliation: Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Hong-Yi
authors.initials: HY
authors.lastname: Gong
authors.affiliation: Department of Industrial Engineering and Management, National Quemoy University, Kinmen, Taiwan.
authors.forename: Tsung-Yin
authors.initials: TY
authors.lastname: Ou
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
authors.forename: Jue-Liang
authors.initials: JL
authors.lastname: Hsu
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan. Electronic address: syhu@mail.npust.edu.tw.
authors.forename: Shao-Yang
authors.initials: SY
authors.lastname: Hu
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques; D002021:Buffers; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D015870:Gene Expression; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D002479:Inclusion Bodies / Q000737:chemistry*; D010473:Perciformes / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D012995:Solubility; D014508:Urea / Q000737:chemistry
chemical_list: D002021:Buffers; D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; D014508:Urea; D013006:Growth Hormone
keywords: Fed-batch culture; Giant grouper; Growth hormone; Recombinant E. coli