title: Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.
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 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. bioyuxw@aliyun.com.|State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. bioyuxw@aliyun.com.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Yu
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.affiliation: Biological Mass Spectrometry Facility, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zheng
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Sha
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. yxu@jiangnan.edu.cn.|State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. yxu@jiangnan.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, USA.|Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
mesh_terms: D002247:Carbon Isotopes / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D007553:Isotope Labeling; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012233:Rhizopus / Q000737:chemistry / Q000201:enzymology*
chemical_list: D002247:Carbon Isotopes; D004220:Disulfides; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D003903:Deuterium; D008049:Lipase
keywords: 2H, 13C, 15N-enriched protein production; Native disulfide bonds; Nuclear magnetic resonance spectroscopy; Rhizopus chinensis lipase