title: Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding.
abstract: Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.
authors.affiliation: Dept. of Molecular Genetics, Centre de Recerca Agrigenomica CRAG (CSIC-IRTA-UAB), Jordi Girona Salgado 18-24, 08034 Barcelona, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Carvajal
authors.forename: Jordi
authors.initials: J
authors.lastname: Gibert
authors.forename: Nefertiti
authors.initials: N
authors.lastname: Campos
authors.forename: Oriol
authors.initials: O
authors.lastname: Lopera
authors.forename: Eduard
authors.initials: E
authors.lastname: Barberà
authors.forename: Jose M
authors.initials: JM
authors.lastname: Torné
authors.forename: Mireya
authors.initials: M
authors.lastname: Santos
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D002479:Inclusion Bodies / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D011503:Transglutaminases