Strategies for Membrane Protein Success: CLR and RAMP1 in P. pastoris through Mono and Bicistronic Lenses

Abstract

Eukaryotic membrane proteins, notably GPCRs, are involved in a broad spectrum of essential cellular functions and have high biological and pharmaceutical interest being central to drug development. However, researchers face many challenges in obtaining in-depth functional and structural information due to their hydrophobic nature and instability outside their native environment. The objective of this thesis was to address challenges in membrane protein research by applying expression optimisation strategies, with the spotlight on the CGRP receptor, comprising CLR and RAMP1, using the P. pastoris yeast system for high-yield production. The CGRP receptor, which is a membrane protein complex, was a suitably challenging target for this study. The interactions within CLR-RAMP1 provided a unique platform to explore co-expression strategies, particularly the 2A viral peptide technology. Through its expression, the research evaluated various strategies, from construct design and signal peptide selection to strain choice and optimal culturing conditions. The study highlighted the successful expression of CLR and RAMP1 in monocistronic constructs and co-expressed via bicistronic constructs in SMD1163 and X33 cells. The co-expression of CLR and RAMP1 in bicistronic constructs showed promising results, but further optimisation is needed to enhance its effective use in yeast. Additionally, deglycosylation analysis highlighted the heterogeneity introduced by posttranslational modifications in membrane protein expression in P. pastoris. In conclusion, this research has made progress in the expression of CLR and RAMP1, contributing valuable insights to the broader field of membrane protein research. The findings and methodologies presented hold potential implications not only for the P. pastoris system but also for drug development and other related fields.