On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

Clark, Lindsay; Dikiy, Igor; Rosenbaum, Daniel M.; Gardner, Kevin H.

doi:10.1007/s10858-018-0204-3

Cited by 34 publications

(30 citation statements)

References 78 publications

(96 reference statements)

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“…While many SSNMR studies quantify the structure and dynamics of membrane proteins within bilayers, the advancement of this technology is often limited by sample availability: the quantity of protein produced per gram of 15 N, 13 C, and/or 2 H enriched material is a major limiting factor for NMR studies. While simpler eukaryotic expression systems such as Pichia pastoris show great promise in high-yield expression of isotopically enriched material, they do not share the simplicity of E. coli expression, and extensive selective or skip labeling strategies are still under development [1][2][3][4]. Thus, there is still much to be gained by improving the expression yield of functional proteins from prokaryotic expression systems.…”

Section: Introductionmentioning

confidence: 99%

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

et al. 2020

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The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

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Section: Introductionmentioning

confidence: 99%

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

et al. 2020

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“…Both solid and solution NMR spectroscopies have been successfully used to probe the structures of membrane proteins, which are normally challenging to crystallize [21,22,23]. Many membrane proteins have been characterized using solution and solid-state NMR spectroscopy [24,25,26].…”

Section: Introductionmentioning

confidence: 99%

Applications of In-Cell NMR in Structural Biology and Drug Discovery

Kang

2019

IJMS

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In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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“…P. pastoris is an ideal host for the production of isotopically labelled proteins given its ability to perform post-translational modifications resulting in natively-folded eukaryotic proteins [66][67][68]. Additionally, there is a large number of established protocols for efficient and inexpensive isotopic labeling of proteins in P. pastoris and its usefulness as the expression host for numerous proteins for crystallographic structures [69][70][71].…”

Section: Introductionmentioning

confidence: 99%

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

et al. 2020

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Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.

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On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

Cited by 34 publications

References 78 publications

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Applications of In-Cell NMR in Structural Biology and Drug Discovery

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

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