Establishing site-specific recombination mediated cassette exchange in Pichia pastoris and development of an optimized process for the production of mouse Tmprss2

Abstract

Structural and biochemical analyses usually require high amounts of highly pure target proteins. Therein, the production of the increasingly demanded complex, difficult to express target proteins often presents a major bottleneck. Hence, the optimization of expression hosts and process strategies is an indispensably important task to pave the way for further studies. The methylotrophic yeast Komagataella phaffii (commonly known as Pichia pastoris) is a widely used expression system for the production of recombinant proteins. It presents an intriguing alternative to the costly mammalian or insect cell culture, as it unites the cost-efficient and simple handling of microorganisms with many of the sophisticated posttranslational processing capabilities of higher eukaryotes. However, the commonly required stable transformation of P. pastoris via homologous recombination results in several drawbacks. These include a limited number of established genomic loci for the integration of genes of interest (GOI). Moreoever, due to the strongly varying expression results of the transformants, extensive expression screens to identify high-producer (“jackpot”) clones are usually required. The first part of this work revolves around the establishment of a recombinase mediated cassette exchange (RMCE) system to optimize the expression system P. pastoris. The specific integration of GOI into a definedly exchangeable genomic cassette of a P. pastoris RMCE master cell line through the enzyme Flp recombinase presents a promising approach to eliminate the expression screening due to comparable productivities of exchanged producer clones. Furthermore, yet uncharacterized genomic loci can be made available for stable protein expression by this system. In this work, the establishment of a P. pastoris RMCE system is shown. The second part of this work deals with the establishment of a method for the secretory production of the ectodomain of the difficult to express transmembrane serine protease mouse Tmprss2 for structural and functional analyses. Tmprss2 plays a key role in influenza A virus infection through proteolytic activation of hemagglutinin H1. The first successful production of both the active form and a mutant form (D343N) of Tmprss2 was achieved in P. pastoris. The expression yields could be significantly improved through several optimizations of the cultivation and purification process.