Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein

A. Ideno, M. Furutani, T. Iwabuchi, T. Iida, Y. Iba, Y. Kurosawa, H. Sakuraba, T. Ohshima, Y. Kawarabayashi, T. Maruyama

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22 Citations (Scopus)

Abstract

Improper protein-folding often results in inclusion-body formation in a protein expression system using Escherichia coli. To express such proteins in the soluble fraction of E. coli cytoplasm, we developed an expression system by fusing the target protein with an archaeal FK506 binding protein (FKBP). It has been reported that an archaeal FKBP from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TcFKBP18), possesses not only peptidyl-prolyl cis-trans isomerase activity, but also chaperone-like activity to enhance the refolding yield of an unfolded protein by suppressing irreversible protein aggregation. To study the effect of this fusion strategy with FKBP on the expression of foreign protein in E. coli, a putative rhodanese (thiosulfate sulfurtransferase) from a hyperthermophilic archaeon and two mouse antibody fragments were used as model target proteins. When they were expressed alone in E. coli, they formed insoluble aggregates. Their genes were designed to be expressed as a fusion protein by connecting them to the C-terminal end of TcFKBP18 with an oligopeptide containing a thrombin cleavage site. By fusing TcFKBP18, the expression of the target protein in the soluble fraction was significantly increased. The percentage of the soluble form in the expressed protein reached 10-28% of the host soluble proteins. After purification and protease digestion of the expressed antibody fragment-TcFKBP18 fusion protein, the cleaved antibody fragment (single-chain Fv) showed specific binding to the antigen in ELISA. This indicated that the expressed antibody fragment properly folded to the active form.

Original languageEnglish
Pages (from-to)99-105
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume64
Issue number1
DOIs
Publication statusPublished - 03-2004

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

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