| Transglutaminases (TGases; EC 2.3.2.13) are protein-glutamine y-glutamyl-transferases, which catalyze an acyl-transfer reaction between the y-carboxyamide groups of peptide- or protein-bound glutamine residues. When e-amino groups of lysine residues in certain proteins are presented as acyl acceptor, the e-(y-glutaminyl) lysine bonds are introduced and the proteins are cross-linked. The structure and characteristics of proteins will be changed and the new function may be produced. Therefore, TGases are widely applied in agriculture, industry, food processing, cosmetics making and pharmaceutical field.The gene of TGase from Bacillus subtilis was cloned and expressed in three different systems. Then TGase was purified by column chromatography. Finally the characteristics and reaction conditions of TGase were studied in this paper.Firstly, the gene was amplified by PCR, cut with restriction enzymes and transformed into the host. By this way, the gene of TGase from Bacillus subtilis was successfully cloned into three different expression systems, which are Bacillus subtilis pUBIS and pUBD, pichia pastoris pPIC9 and Escherichia coli pET32a(+). The results were different in three systems. In Bacillus subtilis, TGase was expressed at low level with the help of enchancer degQ, but the expressed protein could purified with difficulty. In Pichia pastoris, a protein band was shown by SDS-PAGE with methanol induction. The expressed protein was larger than the theoretical one and the biological activity could not be detected. In Escherichia coli, TGase was expressed in the form of soluble and active enzyme. TGase would be retained the biological activity even when there was another protein (thioredoxin in this paper) was fused at its N teminal.TGase was expressed in Escherichia coli in the end by contrast with another two systems. While expression conditions such as temperature and IPTG concentration were optimized,Trx-TGase fusion protein was expressed in the supernatant with a level up to 5 percent of the total bacterial soluble proteins. The bacteria with IPTG induction were collected and lysed by ultrasonic. Trx-TGase fusion protein was isolated and purified by metal chelate affinity chromatography and 'Superdex 75 size exclusion chromatography from the supernatant of the bacterial lysate.The characteristics of Trx-TGase and TGase monomer were compared. As the results, both Trx-TGase and TGase monomer had a same functional role and Trx-TGase had better stability than TGase monomer. Finally, the reaction conditions of Trx-TGase were studied in this paper instead of TGase monomer. The amount of polymerized BSA was increased with more Trx-TGase fusion protein. Almost all BSA could be cross-linked into polymers after 20 hours. Furthermore, Trx-TGase could catalyze lysozyme, PEG amine or other proteins to produce the new products. On the temperature stability, Trx-TGase could be stored in 4℃ with no degradation and no activity loss. When stored in 37℃, Trx-TGase were easily degraded but the activity of polymerizing proteins seemed not to be decreased. When some chemicals were taken into consideration for the influence of Trx-TGase biological activity, some reduction agents such as dithiothreitol and beta-mercaptoethanol could promote to increase the TGase activity, while some with primary amine would inhibit the activity.In conclusion, the gene of TGase from Bacillus subtilis was cloned and expressed in Escherichia coli successfully, and then TGase had been purified by metal-chelating chromatography and size exclusion chromatography. The characteristics of TGase were studied with the substrate of BSA. All the results were very valuable for further studies on the TGase characteristics and application.
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