Optimization of the expression of genes encoding poly [3-hydroxyalkanoate] synthase from pseudomonas aeruginosa PTCC 1310 in escherichia coli

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates [PHAs]. To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa [P.aeruginosa] PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli [E.coli] BL21 [DE3] cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37°C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes

Comparison of epothilone and taxol binding in yeast tubulin using molecular modeling

Microtubules are unique cytoskeletal structures that have structural subunits of alphabeta tubulin. Taxol is a typical microtubule stabilizing drug. The epothilones are other natural products with similar mechanism of action totaxol. Despite the highly conserved nature of beta-tubulin, some organism like Saccharomyces cerevesia is resistance to taxol, but sensitive to epothilones. In order to find differences in sensitivity of yeast tubulin to these molecules, we investigated binding mode of the taxol and epothilone A to yeast tubulin using molecular modeling. The multiple sequence alignment of beta-tubulin of different species was performed using ClustalW2. Protein structure of yeast beta-tubulin was constructed with Swiss Model 8.05 by using 1TVK. Modeled tubulin was superimposed with PyMol on 1JFF for comparison of three-dimensional structure of two proteins. Our results showed that one of the most interesting differences in binding mode of these molecules is residue 227. The His227 in bovine makes a hydrogen bond by means of its delta-nitrogen with epothilone A and by means of its epsilon-nitrogen with taxol. The Asn227 of yeast can play role of the delta-nitrogen of imidazole ring of H227, but not of epsilon-nitrogen of it. So yeast tubulin in contrast to taxol can interact with epothilone A. Due to conservation of essential residues for binding [T274, R282 and Q292], epothilone A in comparison with taxol can tolerate the interchange in the binding pocket [R276I]. Our findings may be of a great aid in the rational design of anti-tumor agents that bind to the taxol binding region of tubulin