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

Abstract

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.

Simultaneous Microcystin Degradation and Microcystis aeruginosa Inhibition with the Single Enzyme Microcystinase A

“…MlrA comprised six transmembrane structures with strong membrane protein properties (Figure S4). This might be an important reason that MlrA did not easily fold correctly and clear overexpression was not obtained, thereby precluding purification of pure proteins in a general expression system. , These intrinsic properties thus remain an important challenge for characterizing MlrA. Maltose-binding protein (MBP) has been reported as an ideal protein tag that is very effective in promoting the solubility of target proteins, reducing their degradation, and is also effective for affinity purification .…”

Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions

“…P. pastoris offers multiple advantages for the expression of human membrane proteins for NMR studies, which include the ability to produce functional proteins with high levels of deuterium incorporation [ 36 ]. A growing number of membrane proteins have been expressed in P. pastoris for NMR experiments in both aqueous solutions and solid state samples, including human aquaporin 1 [ 37 ], human aquaporin 2 [ 38 ], and fungal rhodopsin from Leptosphaeria maculans [ 39 ]. Initial NMR data with the human GPCRs CB1 and OX2 also appear to be promising [ 24 ].…”

Cloning and Overexpressing Membrane Proteins Using Pichia pastoris (Komagataella phaffii)

“…Thanks to its special physiology and low operating cost, P. pastoris is also one of the very few organisms adapted to the production of isotopically labeled recombinant proteins for their fine structural and dynamic characterizations by NMR spectroscopies (Wood & Komives, 1999; Whittaker & Whittaker, 2005). Several of these production strategies have been recently transposed and adapted to the production of IMPs, either uniformly or selectively labeled (Barret et al., 2022; Clark et al., 2018; Munro et al., 2020).…”