Preparation of protein samples for NMR structure, function, and small-molecule screening studies

Area of Science:

• Structural Biology
• Biophysics
• Genomics

Background:

• High-quality protein samples are crucial for Nuclear Magnetic Resonance (NMR) studies.
• Advancements in NMR hardware necessitate parallel improvements in protein sample production.
• The Northeast Structural Genomics Consortium (NESGC) developed a platform to address these needs.

Purpose of the Study:

• To describe recent advances in producing high-quality protein samples for NMR studies.
• To outline the NESGC's high-throughput protein production platform and strategies.
• To detail the synergistic use of new protein production methods with advanced NMR hardware.

Main Methods:

• High-throughput cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems.
• Isotope enrichment in minimal media for enhanced NMR signal.
• Utilizing 96-well ligation-independent cloning, analytical expression systems, parallel fermentation, and streamlined purification protocols.

Main Results:

• Over 17,500 protein open reading frames cloned into >28,000 constructs.
• Nearly 5000 proteins purified to homogeneity in milligram quantities.
• Over 950 new protein structures, including >400 NMR structures, deposited in the Protein Data Bank.

Conclusions:

• The NESGC pipeline effectively produces isotope-enriched protein samples of prokaryotic and eukaryotic origin.
• Advanced automated and parallel technologies are suitable for structural biology, proteomics, and genomics research.
• The described platform significantly contributes to structural genomics initiatives and protein structure determination.