Patricia Carvajal and Jordi Gibert contributed equally to this work
1520-6033/asset/BTPR_left.gif?v=1&s=a5be2ff5a0fe6ccdaad74a6d128c142e5f71a8da)
1520-6033/asset/BTPR_right.gif?v=1&s=992d113a3e61fc9d3571812359165df86cf830e4)
Cell Culture and Tissue Engineering
Activity of maize transglutaminase overexpressed in escherichia coli inclusion bodies: An alternative to protein refolding
Article first published online: 7 JAN 2011
DOI: 10.1002/btpr.538
Copyright © 2011 American Institute of Chemical Engineers (AIChE)
Additional Information
How to Cite
Carvajal, P., Gibert, J., Campos, N., Lopera, O., Barberà, E., Torné, J. M. and Santos, M. (2011), Activity of maize transglutaminase overexpressed in escherichia coli inclusion bodies: An alternative to protein refolding. Biotechnol Progress, 27: 232–240. doi: 10.1002/btpr.538
- †
Patricia Carvajal and Jordi Gibert contributed equally to this work
Publication History
- Issue published online: 10 FEB 2011
- Article first published online: 7 JAN 2011
- Accepted manuscript online: 12 NOV 2010 10:52AM EST
- Manuscript Revised: 21 SEP 2010
- Manuscript Received: 21 JUN 2010
- Abstract
- Article
- References
- Cited By
Keywords:
- E. coli protein overexpression;
- inclusion bodies fraction;
- enzymatic activity;
- nondenaturant solubilization;
- maize transglutaminase
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. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011