Purification and in vitro refolding of maize chloroplast transglutaminase over-expressed in Escherichia coli

Abstract

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.

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase

“…The reaction was stopped after 2 h with 1% SDS and read at 405 nm in a Multiskan microtitre reader plate (Thermo LabSystems, Beverly, MA, USA). A standard curve to calculate the amount of recombinant protein was made by plating purified chlTGZ protein (Carvajal-Vallejos et al 2007). The amount of purified chlTGZ was determined by the Bradford protein assay following the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin as the standard.…”

“…The chlTGZ over-expressed protein, visualized by immunolocalization using AbTGZ4 (Carvajal-Vallejos et al 2007), appears as a high number of spots within the IBs of PG and Y chloroplasts (Fig. 4c).…”

Amyloid-Like Protein Inclusions in Tobacco Transgenic Plants

“…For protein expression, transformed (pET28-TGZ) E. coli BL21colonies were grown in LB medium containing 30 µg kanamycin/ml to an OD 600 of 0.4 induced for 3 h with 0.4 mM IPTG, and finally harvested by centrifugation [18]. Intracellular recombinant proteins were released with CelLytic BII reagent (Sigma).…”

“…In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene ( tgz ) has been cloned to date (Patent number WO03102128) [17]. Variants of this TGase have been expressed recombinantly in Escherichia coli [18] and tgz- transplastomic tobacco plants engineered [19]. Here we use Th-T and CR binding, Fourier Transformed Infrared Spectroscopy (FT-IR) and Transmission Electronic Microscopy (TEM) to study the conformational properties of the protein deposits formed by maize transglutaminase (TGZ) in vitro and in the chloroplasts of transplastomic plants, demonstrating that in both cases they exhibit characteristic amyloid features.…”

Transglutaminase is Involved in the Remodeling of Tobacco Thylakoids

“…An important step for the elucidation of the plastidal TGase role in plants was the isolation for the first time in plants of two related complementary maize DNA clones, tgz15 and tgz21, encoding active maize TGase (Torné et al 2002;Villalobos et al 2004). Interestingly, their expression is dependent on the duration of light exposure, indicating a role for adaptation in different light environmental conditions including natural habitats (Pintó-Marijuan et al 2007;Carvajal et al 2007Carvajal et al -2011. Proteomic studies indicates that plastidial maize TGase is a peripheral thylakoid protein forming part of a specific PSII protein complex which includes LHCII, ATPase and PsbS proteins, its expression pattern changing according to chloroplast developmental stage and light regime (Campos et al 2010).…”