Abedi Daryoush, Beheshti Maryam, Najafabadi Ali Jahanian, Sadeghi Hamid Mir Mohammad, Akbari Vajihe
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center School of Pharmacy, Isfahan University of Medical Science, Isfahan, Iran.
Avicenna J Med Biotechnol. 2012 Jan;4(1):47-51.
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.
多年来，塑料的使用使固体废物的处理问题变得复杂。减少塑料垃圾的一种策略是使用可生物降解的塑料。这类塑料中的一组是聚羟基脂肪酸酯（PHA）。迄今为止，已知有超过250种不同的微生物能够合成并积累PHA。大多数假单胞菌菌株能够积累中链长度PHA（mcl-PHA）。在先前的研究中，在铜绿假单胞菌（P.aeruginosa）PTCC 1310中鉴定并克隆了phaC1和phaC2基因。本研究的目的是表达这些基因并优化其表达条件。从载体pTZPHAC1和pTZPHAC2获得的插入片段被亚克隆到pET15b表达载体中。用重组质粒转化感受态大肠杆菌（E.coli）BL21（DE3）细胞后，使用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。通过改变表达条件，如IPTG浓度、与IPTG孵育的时间和温度，对这些酶的表达条件进行了优化，并使用适当的统计分析对所得结果进行了比较。用IPTG诱导PHA合酶基因，并观察和纯化表达的62 kDa蛋白。通过改变表达条件，1 mM IPTG、37°C和2小时孵育在大肠杆菌细胞中提供了最高水平的蛋白质产量。这些结果表明，PhaC基因的诱导条件会影响PHA合酶的表达。
