Secretory production of human leptin in Escherichia coli.
Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.
Jeong
K J
KJ
Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea.
Lee
S Y
SY
D001407:Bacillus / Q000201:enzymology; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D020738:Leptin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism
D020738:Leptin; D011994:Recombinant Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases; D019704:Protein Disulfide-Isomerases