title: 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.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Shih
authors.affiliation: Institute of Biological Chemistry and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors.forename: Chi-Chi
authors.initials: CC
authors.lastname: Chou
authors.affiliation: Institute of Biological Chemistry and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors.forename: Yi-Ling
authors.initials: YL
authors.lastname: Chen
authors.affiliation: Institute of Biological Chemistry and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors.forename: Kai-Fa
authors.initials: KF
authors.lastname: Huang
authors.affiliation: Institute of Biological Chemistry and Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan.
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D002465:Cell Movement; D002853:Chromatography, Liquid; D003500:Cyclization; D010450:Endopeptidases / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018945:Monocyte Chemoattractant Proteins / Q000378:metabolism; D036103:Nanotechnology; D059748:Proteolysis; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D018945:Monocyte Chemoattractant Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D010450:Endopeptidases; C099488:TEV protease; D011761:Pyrrolidonecarboxylic Acid
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