title: Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.
abstract: Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Minh Tan
authors.initials: MT
authors.lastname: Nguyen
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Thu Trang
authors.initials: TT
authors.lastname: Thi Vu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Song
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Seon-Ha
authors.initials: SH
authors.lastname: Chong
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Boram
authors.initials: B
authors.lastname: Jeong
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Han-Bong
authors.initials: HB
authors.lastname: Ryu
authors.affiliation: Bio-FD&C Co. Ltd., Incheon, Korea.
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Moh
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul, Korea.
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D058619:Maltose-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011387:Prokaryotic Cells / Q000187:drug effects / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases