cloning and expression of an indigenous mesophile lipase and evaluation of bacillus codon translation in pichia pastoris under control of two different promoters

نویسندگان

fathollah ahmadpour

bagher yakhchali

seyed safa ali fatemi

ali asghar karkhane

چکیده

lipases are versatile biocatalysts with a wide range of application in food, dairy, leather, paper, pharmaceutical and detergent industries.  in this study, mesophilic lipase gene from an indigenous bacillus pumilus f3 that already its gene had been sequenced and  identified, was cloned and expressed in methylotrophic yeast pichia pastoris and codon translation of lipase gene was evaluated in p. pastoris under control of two different promoters (alcohol oxidase (aox1) and glyceraldehid phosphate dehydrogenase(gapdh) promoters). in addition, the expression conditions of recombinant lipase f3 was optimized in p.  pastoris expression system using bmmy medium at ph=3 ,26ºc in 0.75% methanol.  the lipase gene of 648bp with natural signal peptid sequence from b. pumilus f3 and the codon optimized lipase gene cloned and expressed in methylotrophic yeast p. pastoris . the lipase gene was excised from the recombinant plasmid with bamhi, ecori enzymes and ligated to the ppic9 and pgap9 linearized with the same enzymes. the recombinant plasmids were confirmed by the pcr and restriction enzyme digestion. the bgl ii linearized ppic9 and pgap9 recombinant plasmids were introduced into the yeast p. pastoris gs115 genom by electroporation and confirmed by pcr. lipase expressing yeast was cultivated in a 250-ml shaking flask containing medium expression. expression of lipase gene was confirmed using p-nitrophenyl palmitate test and sds-page. codon optimized lipase produced as well as native gene and lipase expression was low in both. also, these results suggest that protein structure is more important than codon preference in expression of proteins such as lipases.

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عنوان ژورنال:
applied biotechnology reports

جلد ۳، شماره ۲، صفحات ۴۱۳-۰

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