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Identifier

000388586

Title

Κλωνοποίσηση, έκφραση και μερικός καθαρισμός πιθανών απακετυλασών χιτίνης από το λεπιδόπτερο Helicoverpa armigera

Alternative Title

Cloning expression and partial purification of putative chitin deacetylases of Helicoverpa armigera

Author

Μαρίνης, Δημήτρης Α.

Thesis advisor

Μπουριώτης, Βασίλης

Reviewer

Βόντας, Ιωάννης
Πετράτος, Κυριάκος

Abstract

In order to develop new approaches to confront insects that affect crops, a number of insects’ enzymes have been targeted. This study is part of a general research effort to evaluate chitin deacetylases as target enzymes for the development of specific inhibitory compounds as novel pest control agents. As a model organism the lepidopteron Helicoverpa armigera, a highly polyphagous species was chosen. The aim of this study is the overexpression, isolation and biochemical characterization of three putative chitin deacetylases (HaCDA1, HaCDA5a, HaCDA5b) from H. armigera. For each gene, two plasmid constructs were made, with and without signal peptide into a plasmid vector (pet26b) suitable for expression in bacterial cells of E. coli. The transformation took place at six different E.coli strains namely BL21 (DE3) pLysS, BL21 star (DE3) pLysS, BL21 Codon Plus (DE3) RipL, C43 (DE3), Shuffle (DE3), BL21 star (DE3), PG-KJE8 BL21. However, the expression levels of the recombinant proteins were extremely low for further isolation and biochemical characterization. Therefore, a eukaryotic microorganism namely Pichia Pastoris was subsequently selected for expressing the three genes encoding for putative polysaccharide deacetylases. Genes hacda1, hacda5a, hacda5b were cloned into the plasmid vector pPICAZalpha suitable for transformation of P.pastoris X33 strain. However, the expression levels of the three proteins did not result in improved yields of the enzymes. Gene synthesis and codon optimization of all three genes and subsequent cloning in Pichia Pastoris took place in order to achieve higher expression levelsof the three proteins.

Language

Greek

Subject

Chitin

Strain X33

Στέλεχος Χ33

Χιτίνη

Issue date

2014-11-21

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses