title: Periplasmic production via the pET expression system of soluble, bioactive human growth hormone.
abstract: A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni(2+) affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.
authors.affiliation: Pharmaceutical Sciences and Pharmacogenomics Graduate Program, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 513 Parnassus Ave., Box 0912, San Francisco, CA 94143, USA. jonathan.sockolosky@ucsf.edu
authors.forename: Jonathan T
authors.initials: JT
authors.lastname: Sockolosky
authors.forename: Francis C
authors.initials: FC
authors.lastname: Szoka
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D019897:Periplasm / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D020349:Surface Plasmon Resonance
chemical_list: D019382:Human Growth Hormone