Dissertation (PhD)--Stellenbosch University, 2005. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50271 |
| Date | 04 1900 |
| Creators | Kolar, Norbert Wilhelm |
| Contributors | Swart, Pieter, Swart, Amanda, Stellenbosch University. Faculty of Science. Department of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 263 p. : ill. |
| Rights | Stellenbosch University |
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