人类寡肽转运蛋白hPepT2（579-664）的原核表达及其性质研究
发布时间：2018-07-24 14:33
【摘要】：营养学研究表明，蛋白质在人体内主要以小肽的形式通过寡肽转运载体的转运被吸收。寡肽转运载体POT家族包含PepT1, PepT2, PHT1和PHT2四个成员,负责大多数二肽、三肽及其类似药物的转运。其中，PepT2在很多器官中都有表达，包括肾脏、大脑、眼睛、乳腺等等。人类寡肽转运蛋白hPepT2包含729个氨基酸残基、12个跨膜区和9跨膜区与10跨膜区之间的一个胞外大环，是一种高亲和性、低容量的寡肽转运载体。氨基酸突变法和蛋白嵌合法研究表明，，人类寡肽转运蛋白hPepT2的1-9跨膜区决定其表现特征，7-9跨膜区为其底物结合区域，最后三个跨膜区仍为未知功能区域。 本文对人类寡肽转运蛋白10-11跨膜区片段hPepT2(579-664)进行了原核表达。通过设计引物对hPepT2的cDNA进行PCR克隆获得目的基因，进而通过限制性内切酶进行双酶切和T4连接酶连接，构建了pET30a(+)-hPepT2(579-664)重组质粒。重组质粒经过测序无氨基酸突变，然后转入E.coli BL21(DE3)pLysS中进行表达。对含表达重组体系的菌液在37℃下，使用工作浓度0.3mM的IPTG进行诱导表达，4h后获得良好的表达效果。菌体沉淀再经洗涤、超声破菌、葡聚糖凝胶G-75柱纯化、透析、冷冻干燥得到目标蛋白。利用紫外可见光谱进一步确定了蛋白的水溶性，利用紫外光谱和荧光光谱研究了其在不同pH缓冲液中的稳定性，发现蛋白在pH7.5时较稳定。 利用固相合成法合成了具有生物活性的十个含有酪氨酸的二肽：酪胺酰甘氨酸、酪胺酰亮氨酸、酪胺酰苯丙氨酸、酪胺酰赖氨酸、酪胺酰精氨酸、酪胺酰酪氨酸、丝氨酰酪氨酸、脯氨酰酪氨酸、缬氨酰酪氨酸、β-丙氨酰酪氨酸，并对合成产物进行了高效液相分析、纯化、ESI-MS表征。利用荧光光谱法研究了4种二肽与人类寡肽转运蛋白hPepT2(579-664)的相互作用，考察了肽浓度、作用时间对相互作用的影响。结果表明，二肽与浓度为2.52mg/ml蛋白相互作用，其最适浓度为3.33×10~(-3)-1.0×10~(-2)mg/ml，此时蛋白质的荧光强度最大，同时二肽可以促使蛋白结构发生改变，增加蛋白的荧光发射强度，随着反应时间的延长逐渐增加，最后趋于平衡。证明人类寡肽转运蛋白hPepT2(579-664)可以结合二肽或与二肽相互作用，二者之间存在相互作用。本研究为研究人类寡肽转运蛋白PepT2的功能和底物结构以及新药的设计提供了基础数据。
[Abstract]:Nutritional studies show that proteins are mainly absorbed in the form of small peptides by the transport of oligoseptide transporters. The POT family of oligoseptide transporters consists of four members, PepT1, PepT2, PHT1 and PHT2, which are responsible for the transport of most dipeptides, tripeptides and their analogues. PepT2 is expressed in many organs, including the kidneys, brain, eyes, mammary glands and so on. Human oligoseptide transporter (hPepT2) contains 729 amino acid residues, 12 transmembrane regions and a large extracellular ring between 9 and 10 transmembrane regions, which is a high affinity and low capacity oligopeptide transport vector. The results of amino acid mutation and protein embedding showed that the 1-9 transmembrane region of human oligopeptide transporter (hPepT2) determined its characteristic transmembrane domain 7-9 as its substrate binding region, and the last three transmembrane regions were still unknown functional regions. The transmembrane region hPepT2 (579-664) of human oligopeptide transporter 10-11 was expressed in prokaryotic expression. The recombinant plasmid of pET30a () -hPepT2 (579-664) was constructed by designing primers for PCR cloning of cDNA of hPepT2, and then double restriction enzyme digestion and T4 ligase ligation were used to construct pET30a () -hPepT2 (579-664) recombinant plasmid. The recombinant plasmid was sequenced without amino acid mutation and then expressed in E.coli BL21 (DE3) pLysS. The expression of 0.3mM was induced by IPTG at 37 鈩
本文编号：2141680
[Abstract]:Nutritional studies show that proteins are mainly absorbed in the form of small peptides by the transport of oligoseptide transporters. The POT family of oligoseptide transporters consists of four members, PepT1, PepT2, PHT1 and PHT2, which are responsible for the transport of most dipeptides, tripeptides and their analogues. PepT2 is expressed in many organs, including the kidneys, brain, eyes, mammary glands and so on. Human oligoseptide transporter (hPepT2) contains 729 amino acid residues, 12 transmembrane regions and a large extracellular ring between 9 and 10 transmembrane regions, which is a high affinity and low capacity oligopeptide transport vector. The results of amino acid mutation and protein embedding showed that the 1-9 transmembrane region of human oligopeptide transporter (hPepT2) determined its characteristic transmembrane domain 7-9 as its substrate binding region, and the last three transmembrane regions were still unknown functional regions. The transmembrane region hPepT2 (579-664) of human oligopeptide transporter 10-11 was expressed in prokaryotic expression. The recombinant plasmid of pET30a () -hPepT2 (579-664) was constructed by designing primers for PCR cloning of cDNA of hPepT2, and then double restriction enzyme digestion and T4 ligase ligation were used to construct pET30a () -hPepT2 (579-664) recombinant plasmid. The recombinant plasmid was sequenced without amino acid mutation and then expressed in E.coli BL21 (DE3) pLysS. The expression of 0.3mM was induced by IPTG at 37 鈩
本文编号：2141680
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