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

• Published in: PloS one 2014-04, Vol.9 (4), p.e94812-e94812
• Main Authors: Shih, Yan-Ping, Chou, Chi-Chi, Chen, Yi-Ling, Huang, Kai-Fa, Wang, Andrew H-J
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Aminoacyltransferases - metabolism; Bacteria; Biology and Life Sciences; Bone density; Cell Movement; Chemokines; Chromatography, Liquid; Cleavage; Cloning; Cloning vectors; Cyclization; Cytotoxicity; Design modifications; Drug development; Drugs; E coli; Endopeptidases - metabolism; Enzymes; Fluorescence; Gene expression; Genetic Vectors - metabolism; Green Fluorescent Proteins - metabolism; Guanylate cyclase; Hormones; Humans; In vivo methods and tests; Mammals; Medicine and Health Sciences; Monocyte Chemoattractant Proteins - metabolism; Monocytes; Nanotechnology; Passengers; Peptides; Physiological aspects; Protease; Proteases; Protein folding; Proteinase; Proteins; Proteolysis; Pyrrolidonecarboxylic Acid - metabolism; Recombinant Fusion Proteins - metabolism; Recombinant proteins; Research and Analysis Methods; Tags; Tandem Mass Spectrometry; Tobacco; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0094812

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.