Abstract
BackgroundRecombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.ResultsHere we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory.ConclusionsThe accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.
Highlights
Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture
We examined factors, including pre-induction cellular biomass, affecting the total yield of recombinant green fluorescent protein (GFP) in P. pastoris [25], and noted the benefits of this as a strategy to improve productivity. While this approach is generally accepted to be a useful way to boost the yields of soluble proteins, it is well established that the accumulation of biomass does not necessarily lead to a correlated increase in membrane protein yield [21] and in the case of G protein-coupled receptors (GPCRs), specific activity is often lower [26]
TM6* should prove to be a useful addition to the matrix of production hosts at the disposal of modern structural biology projects, especially for challenging targets such as membrane proteins: we suggest that transfer of expression plasmids from wildtype strains into TM6* is a simple way to at least double the yield of a range of recombinant proteins
Summary
Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. We examined factors, including pre-induction cellular biomass, affecting the total yield of recombinant green fluorescent protein (GFP) in P. pastoris [25], and noted the benefits of this as a strategy to improve productivity. While this approach is generally accepted to be a useful way to boost the yields of soluble proteins, it is well established that the accumulation of biomass does not necessarily lead to a correlated increase in membrane protein yield [21] and in the case of G protein-coupled receptors (GPCRs), specific activity is often lower [26]. Medium cell density fermentation procedures for GPCR expression in P. pastoris have been suggested to be preferable to ones where biomass yields are maximised [26]
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