Overexpression of Membrane Proteins Using <i>Pichia pastoris</i>

Bornert, Olivier; Alkhalfioui, Fatima; Logez, Christel; Wagner, Renaud

doi:10.1002/0471140864.ps2902s67

Cited by 11 publications

(17 citation statements)

References 45 publications

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“…These factors make yeast an inexpensive and efficient alternative to prokaryotic expression systems for production of membrane proteins. Saccharomyces cerevisiae and Pichia Pastoris (Pichia) are the most commonly used species for expression of membrane proteins (Bornert et al 2012;Joubert et al 2010). Schizosaccharomyce pombe is less frequently used, but sometimes outperforms other yeast species in expression of mammalian membrane proteins (Sander et al 1994b;Takegawa et al 2009).…”

Section: Yeastmentioning

confidence: 99%

Current strategies for protein production and purification enabling membrane protein structural biology

Pandey

Shin

Patterson

et al. 2016

Biochem. Cell Biol.

105

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Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryoelectron microscopy (cryo-EM).

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Section: Yeastmentioning

confidence: 99%

Current strategies for protein production and purification enabling membrane protein structural biology

Pandey

Shin

Patterson

et al. 2016

Biochem. Cell Biol.

105

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“…Human membrane proteins. The methylotrophic yeast P. pastoris has been used to produce crystallization-grade proteins for several MPs, from which high-resolution 3D structures have been determined [7,29,70]. In fact, P. pastoris is able to produce proteins with all kinds of membranespanning topologies, including enzymes, aquaporins, and ion channels [7].…”

Section: Biosynthesis Strategies For Protein Production In Pichia Pasmentioning

confidence: 99%

“…The methylotrophic yeast P. pastoris has been used to produce crystallization-grade proteins for several MPs, from which high-resolution 3D structures have been determined [7,29,70]. In fact, P. pastoris is able to produce proteins with all kinds of membranespanning topologies, including enzymes, aquaporins, and ion channels [7]. As highlighted previously by Ramón and Marin [70], the production of functional, properly folded and inserted MPs is often achieved by using alternative approaches that are almost always specific for each target MP.…”

Section: Biosynthesis Strategies For Protein Production In Pichia Pasmentioning

confidence: 99%

“…The use of the mammalian Kozak sequence, optimization of codon usage, coexpression of MPs with the Hac1p, Nterminal fusion of signal sequences to the MP, lower temperature cultivation, and the addition of chemical chaperones such as DMSO are some of the successful strategies that have been employed to improve MP expression in P. pastoris [70]. Moreover, a set of guidelines and instructions with several tips to overexpress MP in the P. pastoris system using GPCR as a model was recently published by Bornert et al [7].…”

Section: Biosynthesis Strategies For Protein Production In Pichia Pasmentioning

confidence: 99%

“…Usually, MPs are produced intracellularly, in which case the membranes need to be isolated. This is accomplished by performing an ultracentrifugation step, typically at 200,000 ×g for 60 min at 4 o C or 30 min at 4 o C after cell lysis [3,4,7].…”

Section: Biosynthesis Strategies For Protein Production In Pichia Pasmentioning

confidence: 99%

See 2 more Smart Citations

Pichia pastoris: A Recombinant Microfactory for Antibodies and Human Membrane Proteins

Gonçalves

Pedro²,

Maia³

et al. 2013

J. Microbiol. Biotechnol.

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During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

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Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

et al. 2020

Self Cite

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Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins (IMPs), for various applications. This chapter details a series of protocols that progress towards the production of IMPs, their extraction and purification in presence of detergents, and their eventual reconstitution in lipid nanoparticles. These P. pastoris-oriented basic procedures can be further optimized in order to deliver IMP samples compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides a number of general guidelines, technical hints and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian IMPs.

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Overexpression of Membrane Proteins Using Pichia pastoris

Cited by 11 publications

References 45 publications

Current strategies for protein production and purification enabling membrane protein structural biology

Current strategies for protein production and purification enabling membrane protein structural biology

Pichia pastoris: A Recombinant Microfactory for Antibodies and Human Membrane Proteins

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

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