Heterologous expression of Chit36 from Trichoderma atroviride in prokaryotic system

Chitinolytic enzymes are involved to break down chitin waste products such as chitin shells of crustaceans and cell walls of fungi and production of chitin oligosaccharides. Ideal catalytic properties of these enzymes make them an attractive target for use in industry or as a biocontrol. In this study to amplify and cloning of chit36 gene from native Iranian isolate, Trichoderma atroviride PTCC5220, Specific primers (chit36pf/chi36r) were designed and then intended to produce in periplasmic form, amplified fragment was cloned into pET26b() vector. The new construct was transformed into E. coli BL21-DE3 bacteria. The results of SDS-PAGE showed no evidence of protein expression in any of the conditions of temperature and different levels of IPTG at different cell fractions. So expression of Chit36 protein with the removal of signal peptide (to produce cytoplasmic form) with histidine sequence at the carboxyl end was planned. Bacteria containing recombinant construct were evaluated at different induction conditions. Despite detection of any band in any induction conditions using SDS-PAGE, low expression of proteins in two colonies were confirmed by Western blot assay. In order to optimize the expression condition, recombinant protein expression levels relative to the total bacterial protein in different induction conditions was evaluated using the quantitative measurements of protein bands on SDS-PAGE gels. The results showed that the best condition to induce expression is at OD600 : 0.3 and 1 mM IPTG and incubation time of 6 hours. In these conditions expression level of recombinant protein relative to total protein was 45.57%.