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

Ashe, Mark P.; Bill, Roslyn M.

doi:10.1002/biot.201000333

Cited by 16 publications

(16 citation statements)

References 52 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Yang

Teymorian

Olivares

et al. 2015

Biotechnology Reports

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Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30 mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1). PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture conditions resulted in approximately eight-fold (75–80 mg/L) increase in extracellular activity compared to PAOXI (8–10 mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Yang

Teymorian

Olivares

et al. 2015

Biotechnology Reports

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Salcedo-Sora

Ward

Biagini

2012

Malar J

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BackgroundCalcium (Ca2+) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented.MethodsThe Plasmodium falciparum Ca2+/H+ antiporter (PfCHA) was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese.ResultsBioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies.DiscussionThe functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent cation/H+ antiporter susceptible to the effects of cation/H+ inhibitors such as KB-R7943. This type of gene expression systems should advance the efforts for the screening of potential inhibitors of this type of divalent cation transporters as part of the malaria drug discovery initiatives and for functional studies in general.ConclusionThe expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca2+ as well as Mg2+ and Mn2+ lend itself to high-throughput and quantitative settings for pharmacological screening and functional studies.

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“…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.…”

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confidence: 99%

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

Marı́n

Blondel

2011

Biotechnology Journal

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

Cited by 16 publications

References 52 publications

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

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

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

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

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