ArticleMicrobial cell factories2016
Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.
Article in Microbial cell factories, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- High-level expression of zearalenone lactonohydrolase ZenG in Pichia pastoris via combinatorial strategy.Archives of microbiology · 2025Article
- Microbial lipases and their industrial applications: a comprehensive review.Microbial cell factories · 2020Review
- Structural Basis by Which the N-Terminal Polypeptide Segment ofBiochemistry · 2019Article
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8 authors.
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Abstract
backgroundIn 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.
resultsThe 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.
conclusionsThese 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.
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