Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR
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 i...
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doaj-477479007b6c4d42ba06b5316f9aafbc2020-11-25T03:29:28ZengMDPI AGBiomolecules2218-273X2020-03-0110343410.3390/biom10030434biom10030434Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMRRachel Munro0Jeffrey de Vlugt1Vladimir Ladizhansky2Leonid S. Brown3Departments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, CanadaDepartments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, CanadaDepartments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, CanadaDepartments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, CanadaSolid-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 <i>Pichia pastoris</i>. 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.https://www.mdpi.com/2218-273X/10/3/434biosynthetic isotope labelingaquaporins<i>pichia pastoris</i>solid-state nmrmembrane proteinspost-translational modification |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Rachel Munro Jeffrey de Vlugt Vladimir Ladizhansky Leonid S. Brown |
spellingShingle |
Rachel Munro Jeffrey de Vlugt Vladimir Ladizhansky Leonid S. Brown Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR Biomolecules biosynthetic isotope labeling aquaporins <i>pichia pastoris</i> solid-state nmr membrane proteins post-translational modification |
author_facet |
Rachel Munro Jeffrey de Vlugt Vladimir Ladizhansky Leonid S. Brown |
author_sort |
Rachel Munro |
title |
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR |
title_short |
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR |
title_full |
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR |
title_fullStr |
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR |
title_full_unstemmed |
Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in <i>Pichia pastoris</i> for Solid-State NMR |
title_sort |
improved protocol for the production of the low-expression eukaryotic membrane protein human aquaporin 2 in <i>pichia pastoris</i> for solid-state nmr |
publisher |
MDPI AG |
series |
Biomolecules |
issn |
2218-273X |
publishDate |
2020-03-01 |
description |
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 <i>Pichia pastoris</i>. 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. |
topic |
biosynthetic isotope labeling aquaporins <i>pichia pastoris</i> solid-state nmr membrane proteins post-translational modification |
url |
https://www.mdpi.com/2218-273X/10/3/434 |
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