Design and optimisation of expression vectors for the synthesis of eukaryotic proteins in plants

Abstract

Recombinant proteins are vital in sectors like medicine, industry, and agriculture. Conventional expression systems face challenges such as the inability of prokaryotic systems to perform eukaryotic post-translational modifications and the high costs of scaling up eukaryotic cell cultures. Plants are scalable and can produce eukaryotic modifications but suffer from low yields and underdeveloped downstream processing methods. This thesis aimed to enhance plant-based production of recombinant proteins, focussing on developing and optimizing plant expression vectors and protein processing procedures to maximize yields. A range of eukaryotic proteins, including those from plants and mammals, varying in size and sub-cellular localisation were expressed to understand how protein complexity affects yield. The goal was to express full-length rat and human P2X7 receptors and two truncated versions of isolated cytoplasmic domains. Using Golden Gate and conventional cloning, several expression constructs were created. These constructs significantly increased eGFP production in plants, up to 199-fold more than initial constructs and 2.2-fold more than the previously published pJL-TRBO vector. By comparing different protein extraction and purification methods an effective downstream processing pipeline was developed, enabling eGFP quantification of up to 20% total soluble protein. Compared to starting constructs, eGFP production was increased 254-fold at the protein level using the best construct when measured by western blot. With methods optimised for eGFP, other target proteins were expressed, extracted, purified, and quantified. This led to the development of several useful expression constructs for plants, enabling detectable production of the 53 kDa isolated cytoplasmic domain of P2X7 in a transient N. benthamiana expression system, although full-length transmembrane protein expression was not detected. Overall, this research successfully developed optimized expression vectors and protein extraction and purification techniques for high yields of poly(His)-tagged cytoplasmic proteins and paves the way for future large-scale research to express more diverse protein targets