Wang Lan-Hsiang, Chmelik Rebecca, Nirenberg Marshall
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1654, USA.
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12721-6. doi: 10.1073/pnas.202461199. Epub 2002 Sep 13.
The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C). The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.
腹侧神经系统缺陷(vnd)/NK-2同源结构域及一些侧翼氨基酸残基在大肠杆菌中表达,纯化至同质,然后将该蛋白共价偶联到琼脂糖凝胶上。含有16个碱基对随机序列的寡脱氧核苷酸通过vnd/NK-2亲和柱层析进行纯化、克隆和测序。vnd/NK-2同源结构域结合位点的共有核苷酸序列显示为T(T/C)AAGTG(G/C)。vnd/NK-2同源结构域与共有序列的表观平衡解离常数(K(D))为1.9×10(-10)M。此外,寡脱氧核苷酸与vnd/NK-2同源结构域结合的竞争结果以及与共有序列仅相差一个碱基对的寡脱氧核苷酸表观K(D)值的测定表明,该序列中四个中心核苷酸AAGT在决定结合亲和力方面起主要作用。