RRC ID |
10902
|
著者 |
Kang SH, Kim DM, Kim HJ, Jun SY, Lee KY, Kim HJ.
|
タイトル |
Cell-free production of aggregation-prone proteins in soluble and active forms.
|
ジャーナル |
Biotechnol Prog
|
Abstract |
We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.
|
巻・号 |
21(5)
|
ページ |
1412-9
|
公開日 |
2005-1-1
|
DOI |
10.1021/bp050087y
|
PMID |
16209544
|
MeSH |
Cell-Free System
Escherichia coli Proteins / chemistry
Escherichia coli Proteins / genetics
Escherichia coli Proteins / metabolism*
Inclusion Bodies / chemistry
Inclusion Bodies / metabolism*
Molecular Chaperones / chemistry
Molecular Chaperones / metabolism*
Protein Disulfide-Isomerases / chemistry
Protein Disulfide-Isomerases / metabolism*
Protein Engineering / methods*
Recombinant Proteins / biosynthesis*
Recombinant Proteins / chemistry
|
IF |
2.334
|
引用数 |
23
|
WOS 分野
|
FOOD SCIENCE & TECHNOLOGY
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
リソース情報 |
ヒト・動物細胞 |
F-36E(RCB0776) |