Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in Vivo

Yan Ping Shih, Chi Chi Chou, Yi Ling Chen, Kai Fa Huang, Andrew H.J. Wang

研究成果: 雜誌貢獻文章同行評審

19 引文 斯高帕斯(Scopus)

摘要

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

原文英語
文章編號e94812
期刊PLoS ONE
9
發行號4
DOIs
出版狀態已發佈 - 4月 14 2014
對外發佈

ASJC Scopus subject areas

  • 生物化學、遺傳與分子生物學 (全部)
  • 農業與生物科學 (全部)
  • 多學科

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