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KMID : 1094720090140030257
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Biotechnology and Bioprocess Engineering
2009 Volume.14 No. 3 p.257 ~ p.265
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Importance of Expression System in the Production of Unnatural Recombinant Proteins in Escherichia coli
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Niraikulam Ayyadurai
Neelamegam Rameshkumar
Soundrarajan Nagasundarapandian
Edwardraja Selvakumar
Park Hyung-Soon
Lee Soo-Jae
Yoo Tae-Hyeon
Yoon Hyung-Don
Lee Sun-Gu
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Abstract
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In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while L-homopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LC-MS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system.
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KEYWORD
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unnatural recombinant protein, expression system, residue specific unnatural amino acid incorporation, L-homopropargylglycine
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