title: Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli.
abstract: High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.
authors.affiliation: Department of Chemical and Biomolecular Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Won Min
authors.initials: WM
authors.lastname: Yoo
authors.forename: Ki Chang
authors.initials: KC
authors.lastname: Keum
authors.forename: Nae Choon
authors.initials: NC
authors.lastname: Yoo
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
authors.forename: Moon-Hee
authors.initials: MH
authors.lastname: Sung
mesh_terms: D000410:Alanine Transaminase / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D051263:D-Alanine Transaminase; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D017353:Gene Deletion; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020738:Leptin / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D020738:Leptin; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase