Pichia pastoris : a viable expression system for steroidogenic cytochrome P450 enzymes

Wepener, Ilse (2005-12)

Thesis (MSc)--Stellenbosch University, 2005.

Thesis

ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17.

AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.

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