Secretory production of human leptin inEscherichia coli

Abstract

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°C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30°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°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°C without the decrease of the amount of leptin produced. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 398–407, 2000.

Engineering Escherichia coli for succinate production from hemicellulose via consolidated bioprocessing

“…Xylanases excretion and the specific enzyme activities were slightly higher when the periplasmic oxidoreductase was co-expressed in E. coli as shown in Table 3. The increased excretion was probably due to the fact that DsbA co-expression increased the soluble protein level in the periplasm [14,33], which further improved the specific enzyme activities of xylanases.…”

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

“…During the translation majority of amino acids are encoded by more than one codons, and each organism carries its own bias in the usage of the 61 available amino acid codons. In every cell, the population of t-RNA closely reflects the codons bias of the mRNA population [58]. When the mRNA of heterologous target genes are over expressed in E. coli, differences in codon usage can block translation due to the demand for one or more t-RNAs that may be rare or lacking in that particular population [59].…”

Aerobic Oxidation of Alcohols with Bifunctional Transition‐Metal Catalysts Bearing C–N Chelate Ligands

“…Overproduction of DsbA in E. coli increases the recovery of recombinant proteins requiring disulfide bonds 132. 133 By coexpressing dsbA in E. coli , groups have successfully expressed α‐amylase/trypsin inhibitor,132 functional T‐cell receptors,134 insulin‐like growth factor‐I (IGF‐I),135 and human leptin 136. The production of proteins with many disulfides is complicated, yet active human tissue‐type plasminogen activator (17 disulfides) has been successfully produced by co‐overproducing DsbA and DsbC 137.…”