dc.contributor.author | Maly, Jan | en |
dc.contributor.author | Crowhurst, Karin A. | en |
dc.date.accessioned | 2018-07-12T19:09:09Z | |
dc.date.available | 2018-07-12T19:09:09Z | |
dc.date.issued | 2012 | en |
dc.identifier.citation | Protein Expression and Purification 84(2), 255-264. (2012) | en |
dc.identifier.issn | 1046-5928 | en |
dc.identifier.uri | http://hdl.handle.net/10211.3/204495 | en |
dc.description.abstract | Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of intramolecular motions in RGS and Gα proteins in their apo and complexed forms, we have successfully developed a protocol for preparing milligram quantities of highly purified, isotopically labeled wild-type human Gαi1 (hGαi1) subunit for NMR studies. High levels of expression in Escherichia coli can be attributed to the use of the SUMO fusion protein system, a bacterial strain that produces rare codons, supplementation of minimal medium with small quantities of isotopically labeled rich medium and a lowered induction temperature. Purification of hGαi1 utilized affinity and size exclusion chromatography, and protein activity was confirmed using fluorescence-based GTP-binding studies. Preliminary NMR analysis of hGαi1 has shown that high-quality spectra can be obtained at near-physiological temperatures, whereas lower temperature spectra display numerous weak and broadened peaks, providing preliminary evidence for widespread μs-ms timescale exchange. In an effort to further optimize the NMR spectra we prepared a truncated form of hGαi1 (hGαi1-Δ31) in which the 31-residue unstructured N-terminus was removed. This resulted in further improvements in spectral quality by eliminating high-intensity peaks that obscured resonances from structured segments of the protein. We plan to use hGαi1-Δ31 in future investigations of protein dynamics by NMR spectroscopy to gain insight into the role of these motions in RGS/Gα binding selectivity. | en |
dc.format.extent | 10 pages | en |
dc.language.iso | en | en |
dc.publisher | Elsevier (Academic Press) | en |
dc.relation.uri | doi.org/10.1016/j.pep.2012.06.003 | en |
dc.subject | Cloning | en |
dc.subject | Molecular | en |
dc.subject | Escherichia coli | en |
dc.subject | GTP-Binding Protein alpha Subunits | en |
dc.subject | Gene Expression | en |
dc.subject | Guanosine Diphosphate | en |
dc.subject | Humans | en |
dc.subject | Magnesium | en |
dc.subject | Nuclear Magnetic Resonance | en |
dc.subject | Biomolecular | en |
dc.subject | Plasmids | en |
dc.subject | Recombinant Fusion Proteins | en |
dc.subject | SUMO-1 Protein | en |
dc.subject | GTP-Binding Protein alpha Subunits | en |
dc.subject | Recombinant Fusion Proteins | en |
dc.subject | SUMO-1 Protein | en |
dc.subject | Guanosine Diphosphate | en |
dc.subject | Magnesium | en |
dc.title | Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein αi1 | en |
dc.type | Article | en |
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