人类红细胞膜带3蛋白C端域Lys892～Phe895在Cl^-转运过程中作用的研究

The Effect of Lys892～Phe895 of C-terminal Domain on The Anion Transport of Band 3 Transmembrane Domain

采用酵母表面展示系统 ,表达带 3蛋白膜段结构域 (Gln4 0 4～Val911)至酵母细胞膜 ,功能研究表明 ,表达后的膜段结构域具有离子转运的活性 ,同时 ,带 3蛋白抑制剂 4 ,4′ 二异硫氰 2 ,2′ 二黄酸芪 (DIDS)能够抑制其离子转运的功能 .利用PCR方法 ,以 pFAST Bac mdb3为模板扩增出带 3蛋白膜段结构域的 4种截断突变体 ,分别去除带 3蛋白C端域后 4个 (Ala90 8～Val911)、 16个 (Asp896～Val911)、 2 0个 (Lys892～Val911)、 32个(Asn880～Val911)氨基酸序列 ,测序后将其克隆至表达载体 pYD1上 ,构建酵母表达质粒 pYD1 Trunc4、pYD1 Trunc16、pYD1 Trunc2 0和 pYD1 Trunc32 ,诱导 4组突变体的蛋白质表达 .然后测定Cl-的转运活性 ,结果发现去除后 2 0个 (Lys892～Val911)氨基酸残基后 ,离子转运活性明显下降 ,而去除后 32个 (Asn880～Val911)后 ,离子转运没有进一步下降 ,说明Lys892～Phe895 4个氨基酸残基在带

Band 3 membrane domain were expressed on yeast membrane surface by pYD1 yeast display system. The expressed membrane domain showed anion transport activity and DIDS could inhibit this function of membrane domain. About 1 500 bp cDNA fragment of truncation mutagenesis of band 3 membrane domain were amplified by PCR, which knockout Ala908～Val911, Asp896～Val911, Lys892～Val911 and Asn880～Val911 of band 3 respectively. After being sequenced, the four gene fragments cloned into Eco RⅠ～ Bam HⅠ sites of pYD1. The recombinant plasmids pYD1 Trunc4/Trunc16/Trunc20/Trunc32 were transformed into yeast EBY100. As control, pYD1 mdb3 was also transformed. After four groups fusion protein were expressed after adding galactose, the Cl - transport activity was measured by using a fluorescent probe SPQ. The result demonstrated that the transport activity of band 3 was decreased when knockout Lys892～Val911 of AE1 C terminal domain, but the transport activity didnt decrease further when knockout Asn880～Val911 of AE1 C terminal domain. These results showed that Lys892～Phe895 amino acids influenced the anion transport of band 3 transmembrane domain.