AbstractsBiology & Animal Science

The ATP-binding cassette (ABC) transporters superfamily is one of the largest integral membrane protein families. These multi-domain proteins are ubiquitiously present in all biological kingdoms. To date, 51 ABC genes have been identified in human genome. Of these, predominantly, overexpression of ABCB1, ABCC1 and ABCG2 is associated with multidrug resistance in tumour cells. ABCG2 is a half transporter with reverse topology, that has a nucleotide binding domain that precedes the transmembrane domain (NBD-TMS6), as compared to ABCB1 and ABCC1 which are full transporters with (TMS6-NBD)2 topology. This unique topological feature of ABCG2 is similar to fungal PDR transporters, in particular to proteins that belong to cluster F. ABC transporters are expressed in miniscule amounts in native tumour cells or in other mammalian expression system. Hence, expression of these proteins in a heterologous host is imperative for biomedical research and for the pharmaceutical industry. Despite the current advanced state of heterologous protein expression technology, preparation of high-quality functional protein samples in sufficient amounts by in vitro approaches is still a major bottleneck for the characterization and structural studies of membrane proteins. Therefore, in this thesis work, the possibility of developing Saccharomyces cerevisiae AD∆ strain as a host model expression system for heterologous expression of human ABC transporters was investigated. Human ABCG2 was used as a model protein throughout this study because of its unique characteristic features and phylogenetic relevance to orthologous proteins in Saccharomyces cerevisiae. Despite a fungal PDR transporter – like topology and close phylogenetic evolution, expression of ABCG2 in S. cerevisiae has yet to yield significant quantities of functional enzyme as host biology can make heterologous expression and correct localisation of this transporter a challenge. To obtain ABCG2 in functionally folded conformation many promising approaches were investigated. In this regard, several ABCG2 mutants or PDR-ABCG2 chimera proteins were created; their expression, localisation and drug efflux activity were evaluated in S. cerevisiae AD∆ strain, which is deleted in 7 major efflux pumps. In addition, localisation and functional activity of S. cerevisiae protein Yol07Cp (putative orthologs of ABCG2) was also investigated. Furthermore, the significance of cholesterol in plasma membrane for localisation of ABCG2 was investigated using an ergosterol deficient strain S. cerevisiae AD∆H∆, which can grow auxotrophically in presence of cholesterol. Along with functional protein expression these approaches emphasized on stable localisation of ABCG2 in plasma membrane. Results show that ABCG2 constructs are expressed in S. cerevisiae AD∆ regardless of presence of affinity or reporter tags. All tested mutant ABCG2 constructs and chimera protein were also expressed. However, none of the protein constructs localised to the plasma membrane. In addition, they were non-functional in…