Abstract
A study describing the 1H, 13C and 15N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of γδ T cells.
Highlights
MethodsThe recombinant Skint-1 DV residues S24 to T141 (without the Immunoglobulin constant domain and transmembrane spanning region) was overexpressed in Escherichia
Introduction cdT cells are a subset of unconventional lymphocytes that are often localised to body surfaces, where they comprise a fraction of intraepithelial lymphocytes (IELs) and are thought to contribute to lymphoid stress surveillance, responding to signs of tissue damage, microbial infection and carcinogenesis (Hayday 2009)
A study describing the 1H, 13C and 15N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of cd T cells
Summary
The recombinant Skint-1 DV residues S24 to T141 (without the Immunoglobulin constant domain and transmembrane spanning region) was overexpressed in Escherichia. The pellet was washed three times in a Triton wash buffer [0.5 % Triton X-100 (v/v), 200 mM NaCl, 10 mM EDTA, 0.01 % Na Azide and 50 mM Tris– HCl pH 8.0]. Skint-1 DV (30 mg) was renatured by using a dilution refolding method consisting of addition of Skint IgV protein drop wise to 1 L refolding buffer containing 5 M urea, 100 mM Tris, 0.4 M L-arginine-HCl, 2 mM EDTA, 0.5 mM oxidised glutathione, 5 mM reduced glutathione and 0.1 mM PMSF, pH 8.3 at 4 °C overnight. Skint-1 DV was at a concentration of 1.4 mM in MES pH 6.5 and 50 mM NaCl. Spin system and sequential assignments were made from BEST 1H, 15NHSQC, CBCA(CO)NH, HNCACB, HNCA, HN(CO)CA, HNCO, HN(CA)CO and standard H(C)CH TOCSY, (H)CCH TOCSY, 15N-edited NOESY-HSQC (smix = 100 ms) and 13C-edited NOESY-HSQC experiments (smix = 100 ms) (Muhandiram and Kay 1994; Schanda et al 2006; Lescop et al 2007). All spectra were processed using NMRPipe (Delaglio et al 1995) and analysed using SPARKY (Goddard TD 2004)
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