Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

Salicaceae Endophytes: Growth Promotion Potential in Rice (Oryza sativa L.) and Maize (Zea mays L.) and Bio-Control of Plant Pathogen

Thesis (Ph.D.)--University of Washington, 2016-08

Investigating the Function of a Small Secreted Protein Family in Physcomitrella Patens

The correct development of multicellular organisms depends upon the perception of signals secreted by cells in order to co-ordinate cell differentiation. The Physcomitrella patens genome encodes many components of potential signaling systems, including putative receptor proteins and putative secreted protein ligands, yet at present little characterization of these proteins has been carried out. We are currently attempting to characterize the expression pattern and function of a family of 6 secreted proteins exhibiting homology to PrsS, the ligand that controls self-incompatibility (SI) in Papaver rhoeas (field poppy). In poppy, PrsS interacts a receptor on the surface of pollen tubes, PrpS causing SI by programmed cell death. Homologues of this protein (SPH – S-Protein Homologues) exist in dicotyledonous plants and bryophytes but not in other plant taxa. We aim to determine spatiotemporal expression differences between these proteins via reporter gene analysis and qPCR of cDNA. In addition we are in the process of creating targeted gene knockouts for all 6 of the genes in P. patens. We are also searching for receptors of PrpS in Physcomitrella using a bioinformatic strategy alongside phage display. In accomplishing this we hope to determine the function of a small novel secreted protein family in Physcomitrella but in addition we also hope to elucidate the function of SPH proteins in Arabidopsis.