DESIGN, SYNTHESIS, AND FUNCTIONAL EXPRESSION OF A GENE FOR CHARYBDOTOXIN, A PEPTIDE BLOCKER OF K+ CHANNELS
DESIGN, SYNTHESIS, AND FUNCTIONAL EXPRESSION OF A GENE FOR CHARYBDOTOXIN, A PEPTIDE BLOCKER OF K+ CHANNELS
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DOI：
10.1073/pnas.88.6.2046
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发表时间：
1991-03-01
影响因子：
11.1
通讯作者：
MILLER, C
中科院分区：
文献类型：
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作者：
PARK, CS;HAUSDORFF, SF;MILLER, C
设计合成了蝎毒K+通道阻滞剂charybdotoxin （CTX）的编码基因，并在大肠杆菌中表达为可切割融合蛋白。一个序列特异性蛋白酶，因子X(A)，被用来切割融合蛋白，从而释放毒素肽。重组毒素被纯化，氧化形成二硫键，并处理形成n端焦谷氨酸。重组CTX在高效液相色谱迁移率、氨基酸组成和n端修饰方面与天然毒液CTX相同。以单一Ca2+激活的K+通道作为检测系统，重组CTX显示出与天然毒液相同的阻断和解离动力学。功能活性CTX基因的合成和高水平表达，为研究毒素-离子通道相互作用的基本机制提供了可能。
A gene encoding charybdotoxin (CTX), a K+ channel blocker from scorpion venom, was designed, synthesized, and expressed as a cleavable fusion protein in Escherichia coli. A sequence-specific protease, factor X(a), was used to cleave the fusion protein and thus release the toxin peptide. The recombinant toxin was purified, oxidized to form disulfide bonds, and treated to form N-terminal pyroglutamate. Recombinant CTX is identical to the native venom CTX with respect to high-performance liquid chromatography mobility, amino acid composition, and N-terminal modification. With single Ca2+-activated K+ channels as an assay system, recombinant CTX shows blocking and dissociation kinetics identical to the native venom toxin. The synthetic gene and high-level expression of functionally active CTX make it possible to study the fundamental mechanism of the toxin-ion channel interaction.