Efficient production of 2H, 13C, 15N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Zhang, Meng; Yu, Xiao-Wei; Swapna, G.; Xiao, Rong; Zheng, Haiyan; Sha, Chong; Xu, Yan; Montelione, Gaetano
• Is Part Of: Microbial cell factories. Volume 15:Issue 1(2016)
• Page Start: 1
• Page End: 12
• Publication Date: 2016-12
• Subjects: Rhizopus chinensis lipase -- 2H, 13C, 15N-enriched protein production -- Native disulfide bonds -- Nuclear magnetic resonance spectroscopy; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-016-0522-7
• Languages: English
• ISSNs: 1475-2859

Abstract

Abstract Background 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 andPichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. TheP. pastoris system can be used to produce13 C, 15 N-enriched and even2 H, 13 C, 15 N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups inP. 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. Results The 33 kDa protein—Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using bothP. pastoris andE. coli BL21trxB (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 of13 C, 15 N-enriched r27RCL produced usingP. pastoris was 1.7 times higher that obtained usingE. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced inP. pastoris exhibit O-glycosylation, while the protein samples produced inE. coli were not glycosylated. The specific activity of r27RCL fromP. pastoris was ~1.4 times higher than that produced inE. coli . Conclusions These data demonstrate efficient production of2 H, 13 C, 15 N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using theE. coli BL21trxB (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 other2 H, 13 C, 15 N-enriched, Ile (δ1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.