Department of Bioengineering and Technology, Faculty of Techno...

Among various in vitro plant culture systems hairy root systems seem to be one of the most appealing methods of recombinant protein production due to their advantages in combining both whole-plant cultivation and suspension cell culture platform This is a report on production and secretion of a recombinant pharmaceutically active protein from hairy roots cultures of Withania somnifera to improve the economic potential of this plant for the production pharmaceutical compounds In this study we selected and synthesized a codon-optimized globular adiponectin gAd gene with a calreticulin signal peptide and cloned the sequence into a plant expression binary vector containing a nptII gene as a selectable marker gene The transgenic hairy roots were produced by Agrobacterium rhizogenes-mediated transformation protocol developed by our group Among ten established nptII positive hairy roots lines six colons significantly accumulated gAd protein in the biomass and extracellular medium The presence of gAd was confirmed by western blot analysis of root extracts The maximum level of hairy root biomass growth rate GR intra- and extracellular gAd expressions were obtained after 25-26 days of culture on MS medium The maximum level of intra- and extracellular gAd proteins were found to be 15 19 g gFW and 215 7 g L respectively which resulted in a significant decrease in the amount of intra- and extracellular withanolide A and withaferin A production The addition of PVP KNO3 and NaCl significantly increased the level of extracellular gAd by approximately 13 folds This improvement could significantly increase the amount of intra- and extracellular withanolide A and withaferin A production too The recombinant gAd produced from W somnifera is functional as proved by induction the phosphorylation of ACC in C2C12 muscle cells as its functional amount was 5 1-fold more than gAd produced from E coli and 45 lower than CHO cells Copyright 2020 Published by Elsevier B V

Department of Bioengineering and Technology, Faculty of Technology, Gauhati University, Guwahati, Assam 781014, India. Electronic address: [email protected].

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Production of pharmaceutical active recombinant globular adiponectin as a secretory protein in Withania Somnifera hairy ...