Optimization of the Expression of Genes Encoding Poly (3-hydroxyalkanoate) Synthase from Pseudomonas aeruginosa PTCC 1310 in Escherichia coli

Abstract

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.

Keywords: PhaC1; PhaC2; Polyhydroxyalkanoate; Protein expression.

Figure 1

Electrophoresis of the expressed phaC1 and phaC2 proteins on 12% SDS-PAGE. 1) Protein marker; 2) Total protein from induced E.coli BL21(DE3) containing pET-phaC1; 3) Total protein from induced E.coli BL21 (DE3) containing pET-phaC1 after incubation with Ni-NTA His Bind resin; 4) Purified his-tagged phaC1 protein formNi-NTA His Bind resin; 5) Total protein from induced E.coli BL21(DE3) containing pET15b without insert; 6) Total protein from induced E.coli BL21(DE3) containing pET15b without insert after incubation with Ni-NTA His Bind resin; 7) Purified sample from induced E. coli BL21 (DE3) containing pET15b without insert; 8: Purified his-tagged phaC2 protein form Ni-NTA His Bind resin; 9) Total protein from induced E.coli BL21(DE3) containing pET-phaC2 after incubation with Ni-NTA His Bind resin

Figure 2

The effect of temperature of incubation with IPTG (°C) and IPTG concentration (mM) on SDS-PAGE band intensity of target protein: Error bars show standard errors of mean. C: IPTG concentration, T: temperature of incubation with IPTG. A) phaC1, the intensity of the band observed in lane 6 was significantly different from lanes 1, 3, 9,10, 11 and 12. B) phaC2, the intensity of the band observed in lane 6 was significantly different from lanes 1, 2, 3, 4, 9, 10, 11 and 12. (ANOVA, post hoc, n=3 and p<0.05)

Figure 3

The effect of the time of incubation with IPTG (h) and IPTG concentration (mM) on SDS-PAGE band intensity of target protein: H: time of incubation with IPTG, C: IPTG concentration. A) PhaC1, the intensity of the band observed in lane 5 was significantly different from lanes 1, 4, 7 and10. B) PhaC2, the intensity of the band observed in lane 5 was significantly different from lanes 3, 7, 10 and 12. (ANOVA, post hoc, n=3 and p<0.05)

Figure 4

Expression of phaC gene as visualized by Nile Red plate assay.1) E.coliBL21(DE3) carrying the phaC2 gene, 2) E.coli BL21(DE3) cells without the phaC2 gene (as a negative control) and 3) P.citranolleise (as a positive control)