Mini-Bioreactor Platform for Membrane Protein Production in Komagataella pastoris.

Membrane proteins (MPs) play vital roles across various cellular functions, biological processes, physiological signaling pathways, and human-related disorders. Considering the clinical relevance of MPs and their application as therapeutic targets, it is crucial to explore highly effective production platforms and purification approaches to ultimately obtain a high-resolution structure of the target. Therefore, it would be possible to gather detailed knowledge on their mechanism of action which will be the basis for the rational design of novel and stronger drugs. Unfortunately, when compared to their soluble counterparts, 3D structures of MPs are really scarce (<2%), mainly due to poorly natural abundance, challenges associated with protein solubility and stability, and difficulties in producing bioactive and properly structural folded targets. These drawbacks could significantly impair the use of MPs as therapeutic targeting and demand efforts to develop tailor-made strategies for their appropriate handling. Therefore, this chapter is focused on describing a detailed and high-throughput procedure for the biosynthesis of MPs using Komagataella pastoris cell cultures as expression system in a mini-bioreactor platform. Additionally, insights on a purification strategy that combines immobilized-metal affinity and ion-exchange chromatography are described to further obtain the target protein with a significant degree of purity.