Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli |
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Authors: | Niraikulam Ayyadurai Rameshkumar Neelamegam Soundrarajan Nagasundarapandian Selvakumar Edwardraja Hyung Soon Park Soo Jae Lee Tae Hyeon Yoo Hyungdon Yoon Sun-Gu Lee |
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Institution: | (1) Department of Chemical and Biochemical Engineering, Pusan National University, Busan, 609-735, Korea;(2) Institute for Environmental Technology and Industry, Pusan National University, Busan, 609-735, Korea;(3) Probiond Co., Ltd., Konkuk University, Seoul, 143-834, Korea;(4) Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA;(5) School of Biotechnology, Yeungnam University, Gyeongsan, 712-749, Korea |
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Abstract: | 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 LCMS/MS that Hpg was incorporated into the methionine
residues of the recombinant proteins produced by the pQE expression system.
The first two authors equally contributed to this work. |
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Keywords: | unnatural recombinant protein expression system residue specific unnatural amino acid incorporation L-homopropargylglycine |
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