Purification of heterologous membrane proteins Cytochrome P450 CYP81A9 and Homogentisate Solanesyltransferase

Abstract

The enzyme homogentisate solanesyltransferase (HST) of Arabidopsis thaliana is a member of the UbiA superfamily of intramembrane prenyltransferases and is naturally found in the plant chloroplast envelope and in the plastid membrane. These enzymes play a key role in prenylation and thus in the biosynthetic pathway of chlorophylls, vitamin E, heme, and various quinone derivatives. HST itself catalyzes a reaction leading to the immediate precursor of plastoquinone. Since plastoquinones act as mobile electron carriers in the light-reaction of plants, inhibition of their biosynthesis results in seedling-lethal phenotypes.The second protein investigated belongs to the family of Cytochromes P450, which is known for metabolizing herbicides into non-toxic compounds. In eukaryotic cells, these are mostly located in the endoplasmic reticulum or the inner mitochondrial membrane. One member of this family is the protein Cytochrome P450 CYP81A9 (referred to as CYP81A9) of Zea mays. As most Cytochrome P450s in plants, it has a substrate specificity for a small number of herbicides, such as classes of sulfonylurea and triketones.In order to gain more detailed knowledge about HST and CYP81A9, and to possible new substrates for them, it is necessary to obtain them in purified form. To enable this follow-up research, a strategy for purifying the membrane proteins was developed.Both, the typically low concentration of membrane proteins in their native environment and the slow growth of plants represent reasons for heterologous production of plant membrane proteins in yeasts.. Pichia pastoris is a methylotrophic yeast widely used in biotechnological applications in both industry and in science to produce recombinant proteins. The relatively simple applicability of genetic manipulation and the stable integration of heterologous genes into the genome makes P. pastoris an attractive producer. Furthermore, it is possible to achieve comparatively high biomass concentration and high abundance of proteins during fermentation of P. pastoris.This thesis focuses on developing a strategy for purification and recovery of the recombinant membrane proteins HST and CYP81A9 from P. pastoris, by optimizing the downstream steps of protein solubilisation and purification, using a combination of detergent-based solubilisation and column chromatography.