AI SearchCloning of fungal chitin deacetylase gene into yeast Pichia pastoris
Call Number: AIT Thesis no. BP-00-10 Material type:
SeriesSeries: Asian Institute of Technology. Thesis ; no. BP-00-10Publication details: Bangkok : Asian Institute of Technology, 2000Description: 82, [2] leavesSubject(s): Online resources: Dissertation note: Thesis (M.Sc.) - Asian Institute of Technology, 2000 Summary: Chitin deacetylase (CDA) gene has been amplified from Colletotrichum lindemuthianum UPS9 DNA and cloned into one of the E.coli-Pichia pastoris shuttle vector (pPlC9K). The sequence analysis of cloned gene showed 98% homology with that of C. /indemuthianum ATCC56676. The gene was cloned into pPIC9K in frame with the asecretion signal factor present in the vector. Furthermore, the gene was transformed into Pichia pastoris expression host in order to obtain overexpressing strains. Two strains of Pichia namely GS115 (fast methanol utilizing, Mut+) and KM71 (slow methanol utilizing, Mut5 ) were selected as a host for the exrpession of CDA gene. Pichia transformants were selected on the basis of Histidine synthesizing phenotype and were screened for transformants carrying multi copy inserts of CDA gene on the basis of degree of resistance to G418 (Geneticine ). Transformants carrying resistance upto 3 mg/ml of G418 were screened out which might carry several copies of cloned CDA gene. The expression of cloned CDA gene in Pichia transformants were studied by analysing culture supernatant on SOS-PAGE for excreted protein expression.
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A thesis submitted in partical fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development
Thesis (M.Sc.) - Asian Institute of Technology, 2000
Chitin deacetylase (CDA) gene has been amplified from Colletotrichum lindemuthianum UPS9 DNA and cloned into one of the E.coli-Pichia pastoris shuttle vector (pPlC9K). The sequence analysis of cloned gene showed 98% homology with that of C. /indemuthianum ATCC56676. The gene was cloned into pPIC9K in frame with the asecretion signal factor present in the vector. Furthermore, the gene was transformed into Pichia pastoris expression host in order to obtain overexpressing strains. Two strains of Pichia namely GS115 (fast methanol utilizing, Mut+) and KM71 (slow methanol utilizing, Mut5 ) were selected as a host for the exrpession of CDA gene. Pichia transformants were selected on the basis of Histidine synthesizing phenotype and were screened for transformants carrying multi copy inserts of CDA gene on the basis of degree of resistance to G418 (Geneticine ). Transformants carrying resistance upto 3 mg/ml of G418 were screened out which might carry several copies of cloned CDA gene. The expression of cloned CDA gene in Pichia transformants were studied by analysing culture supernatant on SOS-PAGE for excreted protein expression.
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