Abstract:

In this study, total RNA was extracted from young leaves of maize. Reverse transcription PCR (RT-PCR) method was used to obtain full-length transglutaminase (TGase) gene. The amplified fragment was sequenced to have 1605 bp. The gene consisted of 535 amino acid residues with a calculated molecular mass of 60.9 kD. It was identical to the published TGase gene (GenBank NO. AJ421525). This gene fragment was cloned into pET-28a expression vector. The pET-28a-TGase was transformed into E. coli Rosetta (DE3) and expressed in E. coli cells with the induction of 1 mmol/L IPTG. According to quantitative analysis, the expression amount of this protein was approximately 15% of total protein. Western-blot analysis confirmed that this protein was a His-tag recombinant protein. After the purification by Ni2+-NTA affinity chromatography, the purified protein exhibited high purity and one single band was shown in SDS-PAGE. The TGase activity was up to 16 U/mg after purification.

Key words: maize, E. coli, transglutaminase, expression, activity