Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

Abstract

Membrane proteins are targeted by over 50 % of marketed pharmaceuticals, and as most membrane proteins are not naturally abundant, they must be produced recombinantly for the structural biology that is a pre-requisite to structure-based drug design. Unfortunately, obtaining high yields of functional, recombinant membrane proteins remains a major bottleneck in contemporary bioscience. Trial-and-error optimisation studies have not (and cannot) reveal mechanistic details of the biology of recombinant protein production, highlighting the need for defined and rational optimisation strategies based upon experimental observation. To this end, we have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cell factories. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yielding cells: paradoxically, reduced protein synthesis favors higher yields of recombinant membrane protein. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

“…Data presented here suggest that the presence of native signal peptides at the N- and C-termini of rLlALP2 interfered with the secretion of rLlALP2 to the extracellular medium. Host cells suffer metabolic stress when the transcription level of the foreign transgene is high and this may lead to a reduced yield of foreign protein [3], [11], [22], [24]. By switching from the strong, tightly regulated promoter P AOX1 to the weaker, constitutive promoter P GAP and optimizing culture conditions, an eight- to ten-fold increase in extracellular expression of rLlALP2 was achieved.…”

“…However, the expression yield of intracellular rLlALP2 was modest (8–10 mg/L) [16]. Several factors influence the expression of foreign proteins, and the limiting step(s) varies with protein, promoter and host strain [3], [7], [22]. Efforts to relieve the rLlALP2 biosynthesis bottleneck(s) in P. pastoris included optimization of transgene copy number (one copy was best), addressing differences in codon bias by gene sequence optimization, and lowering the cultivation temperature to facilitate proper protein folding; these optimization strategies lead to a modest increase in intracellular expression (25–30 mg/L) [45].…”

“…NP_001001470) signal peptide has also been reported to successfully drive secretion of proteins to the extracellular medium [21], [19]. However, determining the optimum secretion signal for maximum secretion of foreign proteins is protein specific and requires trial-and-error experiments [3], [7], [22].…”

A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter

“…Additionally, recent advances seeking to overcome the difficulties intrinsic to the expression of foreign proteins in yeast, particularly membrane proteins, have rendered transgenic strains with altered ribosomal content that seems to tolerate and much improve heterologous gene overexpression [56,57]. The successful expression of PfCHA in S. cerevisiae and its localization to the yeast vacuole permits a very tractable system for further functional and pharmacological studies as exemplified here in multi-well whole-cell cation transport and inhibitory assays.…”

Editorial: In vivo protein folding – at the crossroad between basic research and biotechnology

“…Recently, in an attempt to obtain a comprehensive view of genetic factors influencing recombinant membrane protein production in the budding yeast Saccharomyces cerevisiae, a comparative transcriptomic analysis of S. cerevisiae cells expressing recombinant membrane protein production at high versus low yield was performed. Ashe and Bill [11] report the identification of a set of genes for which modulating their activity led to high-yielding yeast strains. These results highlight potential bottlenecks at the folding or translocation stages of protein production.…”