title: From gene to HSQC in under five hours: high-throughput NMR proteomics.
abstract: A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.
authors.affiliation: Invitrogen Corporation, 1610 Faraday Avenue, Carlsbad, California 92008, USA. ajshaka@uci.edu
authors.forename: Shiranthi
authors.initials: S
authors.lastname: Keppetipola
authors.forename: Wieslaw
authors.initials: W
authors.lastname: Kudlicki
authors.forename: Bao D
authors.initials: BD
authors.lastname: Nguyen
authors.forename: Xi
authors.initials: X
authors.lastname: Meng
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Donovan
authors.forename: A J
authors.initials: AJ
authors.lastname: Shaka