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.
Subject(s)
Escherichia coli/genetics , Inclusion Bodies/metabolism , Protein Folding , Transglutaminases/metabolism , Zea mays/enzymology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Microscopy, Electron, Transmission , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transglutaminases/geneticsABSTRACT
In contrast to mammalian transglutaminases (TGs), plant members of the superfamily are poorly characterized. In order to produce pure and active TG for its functional and structural studies, variants of maize chloroplast transglutaminase (TGZ, Patent WWO03102128) were sub-cloned into a pET28 vector and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were present mainly as insoluble inclusion bodies. The TGZ4p variant with four B-type repeats (M(r) approximately 55 kDa), was affinity purified from urea-solubilized inclusion bodies. TGZ4p was refolded by rapid dilution in a Ca(2+)- and guanidine-containing buffer. Active TGZ4p shows the general catalytic characteristics described for other TGs.
