Zhang Meng, Yu Xiao-Wei, Swapna G V T, Xiao Rong, Zheng Haiyan, Sha Chong, Xu Yan, Montelione Gaetano T
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
Microb Cell Fact. 2016 Jul 13;15(1):123. doi: 10.1186/s12934-016-0522-7.
In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds.
The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli.
These data demonstrate efficient production of (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using the E. coli BL21 trxB (DE3) system. For certain NMR studies, particularly efforts for resonance assignments, structural studies, and dynamic studies, E. coli provides a cost-effective system for producing isotope-enriched RCL. It should also be potential for producing other (2)H, (13)C, (15)N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.
为了使用最现代的核磁共振光谱方法研究蛋白质结构和动力学，富含同位素的蛋白质样品至关重要。特别是对于较大的蛋白质（>20 kDa），需要全氘代且异亮氨酸（δ1）、亮氨酸和缬氨酸甲基质子化的蛋白质样品来抑制核弛豫，以提供更高的光谱质量，从而获得蛋白质结构和动力学研究所需的关键主链和侧链共振归属。大肠杆菌和毕赤酵母是用于生产富含同位素的重组蛋白质样品以进行核磁共振研究的两种最常用表达系统。毕赤酵母系统可用于生产富含（13）C、（15）N甚至富含（2）H、（13）C、（15）N的蛋白质样品，但在毕赤酵母中生产具有异亮氨酸（δ1）、亮氨酸和缬氨酸甲基质子化基团的全氘代蛋白质的有效方法仍然不可用。糖基化异质性也给核磁共振研究带来了挑战。大肠杆菌表达系统在过量表达全氘代且异亮氨酸（δ1）、亮氨酸、缬氨酸甲基质子化的蛋白质样品方面效率很高，但在生产具有天然二硫键的分泌型真核蛋白质方面通常不成功。
使用毕赤酵母和大肠杆菌BL21 trxB（DE3）系统生产了33 kDa的蛋白质——华根霉脂肪酶（RCL），这是一种重要的工业酶。两个系统生产的样品均表现出相同的天然二硫键形成和相似的二维核磁共振光谱，表明天然蛋白质折叠相似。使用毕赤酵母生产的富含（13）C、（15）N的r27RCL产量比使用大肠杆菌生产的产量高1.7倍，而异位素标记效率低约15%。在毕赤酵母中生产的蛋白质样品表现出O-糖基化，而在大肠杆菌中生产的蛋白质样品未发生糖基化。来自毕赤酵母的r27RCL的比活性比在大肠杆菌中生产的高约1.4倍。
这些数据表明使用大肠杆菌BL21 trxB（DE3）系统能够高效生产具有天然二硫键的富含（2）H、（13）C、（15）N、异亮氨酸（δ1）、亮氨酸、缬氨酸甲基质子化的真核蛋白质r27RCL。对于某些核磁共振研究，特别是共振归属、结构研究和动力学研究，大肠杆菌为生产富含同位素的RCL提供了一种经济高效的系统。它也有潜力生产其他具有天然二硫键的富含（2）H、（13）C、（15）N、异亮氨酸（δ1）、亮氨酸、缬氨酸甲基质子化的真核蛋白质。
