An investigation of heterologous expression of human steroidogenic cytochromes P450 in yeasts

Kolar, Norbert Wilhelm (2005-04)

Dissertation (PhD)--Stellenbosch University, 2005.

Thesis

ENGLISH ABSTRACT: This study: 1. Compares various heterologous expression systems for high-level expression of cytochromes P450. Limitations of the existing cytochromes P450 expression systems are discussed and possibilities to improve the expression yields of human steroidogenic enzymes, are suggested. In addition the potential applications of human steroidogenic cytochromes P450 expressed in Pichia pastoris are illustrated. 2. Describes the cloning and extracellular expression of a recombinant full-length human cytochrome P450 17a-hydroxylase (p45017a) enzyme in Saccharomyces cerevisiae. After the optimisation of expression conditions, it was shown that this system is not suitable for the expression of full-length human P45017a. 3. Describes the cloning and extracellular expression of the full-length human cytochromes P45017a, aromatase, bs and truncated human cytochrome P45017a in P. pastoris. The limitations using P. pastoris as an export system for expressed P450 enzymes were pointed out. 4. Describes the cloning and intracellular expression of the full-length human cytochrome P45017a in P. pastoris as well as the functional expression of human P45017a in P. pastoris, showing progesterone conversion to 17ahydroxyprogesterone and 16a-hydroxyprogesterone in vivo, for the first time. 5. Evaluates developed methods for the preparation of mierosomes from P. pastoris expressing human P45017a and the spectral characterisation of detergent solubilised human P45017a. 6. Describes the development of protocols for the purification of human P45017a from P. pastoris microsomes.

AFRIKAANSE OPSOMMING: Hierdie ondersoek: 1. Vergelyk verskillende heterologiese proteïen uitdrukkings-sisteme vir die preperatiewe produksie van sitochrome P450. Die tekortkomings van bestaande sitochroom P450-uitdrukkings-sisteme word bespreek en moontlikhede om die opbrengs van menslike steroïedogeniese ensieme te verbeter word voorgestel. Die potensiële aanwendings van menslike steroïedogeniese sitochrome P450, wat in Pichia pas/oris uitgedruk word, word ook geïllustreer. 2. Beskryf die klonering en ekstrasellulêre uitdrukking van die rekombinante vollengte menslike sitochroom P45017a-hidroksilase (P45017a) ensiem in Saccharomyces cerevisiae. Na optimisering van die kondisies vir die uitdrukking kon aangetoon word dat hierdie sisteem nie geskik is vir die uitdruk en sekresie van vollengte menslike P45017a nie. 3. Beskryf die klonering en ekstrasellulêre uitdrukking van die vollengte menslike sitochrome P45017a, aromatase, b5 en verkorte menslike sitochroom P45017a in P. pas/oris. Die beperkinge van P. pas/oris as 'n uitvoersisteem vir die uitdrukking van P450 ensieme word bespreek. 4. Besryf die klonering en intrasellulêre ekspressie van die vollengte menslike sitochroom P450 17a. Die funksionele ekspressie van menslike sitochroom P450 17a in P. pas/oris is vir die eerste keer gekarakteriseer. 5. Evalueer die ontwikkelde metodes vir die voorbereiding van mikrosome van P. pas/oris wat menslike P45017a uitdruk en karakteriseer die detergent opgelosbare menslike P45017a t.o.v. spektroskopiese eienskappe. 6. Beskryf die ontwikkelling van protokolle vir die suiwering van die uitgedrukte menslike P45017a vanuit P. pas/oris mikrosome.

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