International Journal of Applied Research
Vol. 2, Issue 3, Part M (2016)
Heterologous expression of Helicoverpa armigera chitinase gene in Pichia pastoris
Esther Shoba R, Manmohan M, Santhosh S, Athmaram TN, Manjulakumari D
Insects’ growth and development is due to the constant deposition and remodelling of the cuticle which is made up of chitin. Initial separation of new cuticle from old cuticle takes place during the apolysis stage of insects’ growth cycle by the accumulation of moulting fluid. Moulting fluid contains high concentrations of chitinolytic and proteolytic enzymes. The chitinolytic and chitin synthase enzymes are responsible for periodic shedding and resynthesizing of chitin containing exoskeleton. Metabolism of chitin, can be a target for selective pest control management. Consequently, insect chitinases and respective genes are gaining importance as a biopesticide. Helicoverpa armigera being a polyphagous pest worldwide, its chitinase gene was considered for expression. The gene of size 1737bp was cloned into E. coli DH5α using pTZ57R/T vector and characterised by PCR and sequenced. Further the chitinase gene was subcloned into yeast transfer vector (pPICZαB) for expression. Linearised recombinant vector containing chitinase gene was introduced into yeast (Pichia pastoris, X-33) by electroporation and the recombinant yeast clones were identified by PCR analysis. The recombinant protein expressed in yeast (yeild of 1.2 mg per litre) was confirmed by SDS- PAGE and Western blotting. These results show that P. pastoris is a convient system for the production of heterologous chitinase that can be used as biopesticide.
How to cite this article:
Esther Shoba R, Manmohan M, Santhosh S, Athmaram TN, Manjulakumari D. Heterologous expression of Helicoverpa armigera chitinase gene in Pichia pastoris. International Journal of Applied Research. 2016; 2(3): 738-745.