Departments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Biomolecules. 2020 Mar 11;10(3):434. doi: 10.3390/biom10030434.
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 . 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.
固态核磁共振（SSNMR）是一种强大的生物物理技术，用于研究膜蛋白；它需要将同位素标记物掺入样品中。这通常是通过在最小培养基中过表达感兴趣的蛋白质来完成的，其中所有（或一些）碳和氮源都是同位素标记的。为了获得具有足够信噪比的多维 NMR 谱，适用于深入分析，需要高产量的同质结构蛋白。一些膜蛋白，如人水通道蛋白 2（hAQP2），表达量较差，这使得以经济的方式生产 SSNMR 样品变得极其困难，因为最小培养基中的生长会给表达宿主带来额外的压力。我们已经为甲基营养酵母中的真核膜蛋白开发了一种优化的生长方案。我们的新生长方案使用山梨醇补充、更高的细胞密度和低温诱导（LT-SEVIN）的组合，将全长、同位素标记的 hAQP2 的产量提高了十倍。通过结合质谱和 SSNMR，我们能够确定蛋白质的翻译后修饰的性质和程度。所得的蛋白质可以功能重建到脂质中，并在二维 SSNMR 光谱分析时产生出色的分辨率和光谱覆盖范围。
