1H, 13C and 15N resonance assignments of the SNT PTB domain in complex with FGFR1 peptide.
1H, 13C and 15N resonance assignments of the SNT PTB domain in complex with FGFR1 peptide.
复制标题
与 FGFR1 肽复合的 SNT PTB 结构域的 1H、13C 和 15N 共振分配。
DOI：
10.1023/a:1026725919008
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发表时间：
2000
影响因子：
2.7
通讯作者：
Zhou,MM
中科院分区：
文献类型：
--
作者：
Dhalluin,C;Yan,KS;Plotnikova,O;Zeng,L;Goldfarb,MP;Zhou,MM
Methods and resultsThe PTB domain of human SNT-1 (residues 11–140) was subcloned into a modified bacterial expression vector pET28b (Novagen) and overexpressed in E. coli BL21 (DE3) cells as a recombinant protein with a C-terminal cleavable hexa-histidine tag. Uniformly 15N-and 15N/13C-labeled proteins were obtained by growing cells in M9 minimal medium containing 15NH4Cl with or without 13C6-glucose (Isotec). Uniformly 15N/13C-labeled and fractionally deuterated protein was prepared using 75% 2H2O in the medium. The protein was expressed mostly in the soluble fraction and purified by affinity chromatography on a nickel-IDA column (Invitrogen). Cleavage of the His6 tag was achieved by treatment with thrombin, leaving an additional four-residue LVPR sequence at the C-terminus from the engineered thrombin site. A protein refolding procedure was used after thrombin treatment, followed by ion-exchange chromatography to ensure proper folding and high purity. The FGFR1 peptide was chemically synthesized on a MilliGen 900 peptide synthesizer (Perkin Elmer) using Fmoc/HBTU chemistry. NMR samples consisted of the SNT-1 PTB domain/FGFR1 peptide (1/1) of approximately 0.5 mM in 100 mM phosphate buffer of pH 6.5, 5 mM DTT-d10 and 0.5 mM EDTA in H2O/2H2O (9/1) or 2H2O. All NMR experiments were conducted at 30◦ C on a Bruker DRX600 or DRX500
