From Gene to HSQC in under Five Hours:High-Throughput NMR Proteomics

摘要

Solution structure determination of recombinant,isotopically enriched proteins by NMR makes demands on protein purity,homogeneity,and absence of contaminants like proteases.With Escherichia coli or baculovirus-mediated insect cell expression,careful protein purification is required for NMR spectroscopy.As the quality of the 2D ~(15)N-~1H heteronuclear single quantum coherence (HSQC) spectrum itself reflects NMR suitability,the time to obtain this spectrum,once the relevant gene is identified,measures potential throughput.A "quick and clean" way to do so would thus be a welcome advance.We show such a protocol using a rapid-fire,cell-free,E.coli-based in vitro expression system.Compatible with purification using histidine fusion tags and immobilized metal ion affinity chromatography,it can also rapidly produce good NMR samples without time-consuming purification.Expressing SUMO-1 in this way,protein quantity was sufficient after a 4 h reaction to obtain 2D ~(15)N-~1H HSQC spectra with a conventional 5 mm HCN triple-resonance probe in under 1 h at 800 MHz.In contrast to previous experience with SUMO-1,in which protein instability was noted,these samples remained stable in the NMR tube for at least 6 months.We used the filter diagonalization method (FDM) to obtain high-resolution 2D NMR spectra.The in vitro expression system,optimized pulse sequence,and FDM combine to provide a good platform for high-throughput NMR proteomics.