Open AccessOptimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli
Author(s) -
Daryoush Abedi,
Fatemeh Moazen,
Vajihe Akbari,
Farnoush Mirzaalian,
Hamid Mir Mohammad Sadeghi
Publication year - 2016
Publication title -
advanced biomedical research
Language(s) - English
Resource type - Journals
ISSN - 2277-9175
DOI - 10.4103/2277-9175.178790
Subject(s) - lac operon , escherichia coli , inducer , recombinant dna , enzyme , plasmid , pseudomonas aeruginosa , expression vector , pseudomonas , biochemistry , microbiology and biotechnology , chemistry , gene , biology , bacteria , genetics
Background: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli.Materials and Methods: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized.Results: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours.Conclusion: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities