Cloning, Large Scale Over-Expression in E-coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter
Cloning, Large Scale Over-Expression in E-coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter
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DOI：
10.1007/s10930-013-9503-4
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发表时间：
2013-08-01
期刊：
影响因子：
3
通讯作者：
Indiveri, Cesare
中科院分区：
文献类型：
--
作者：
Galluccio, Michele;Pingitore, Piero;Indiveri, Cesare
利用大肠杆菌表达系统首次获得了人LAT 1转运蛋白的高效表达。杆菌从HEK 293细胞中扩增hLAT 1 cDNA，并将其克隆到pH 6 EX 3载体中。用构建的pH 6 EX 3 - 6 His-hLAT 1在大肠杆菌Rosetta（DE 3）pLysS菌株中表达6 His-hLAT 1蛋白。杆菌在28 A ℃下用IPTG诱导后8h检测到最高水平的表达。将表达的蛋白质收集在细胞裂解物的不溶性部分中。经SDS-PAGE分析，该多肽的表观分子量为40 kDa。经肌氨酰增溶和尿素变性后，用Ni ~（2+）螯合亲和层析法纯化了N端带有6 His标签的蛋白，并用抗His抗体进行了鉴定。纯化后过表达蛋白的产量为3.5mg/L（细胞培养物）。从Imagene质粒扩增的人CD 98 cDNA克隆到pGEX-4 T1中。构建的pGEX-4 T1-hCD 98在大肠杆菌Rosetta（DE 3）pLysS菌株中表达GST-hCD 98蛋白。杆菌在这种情况下，在28 ℃下用IPTG诱导后4小时检测到最高水平的表达。表达的蛋白质在细胞裂解物的可溶性部分中积累。基于SDS-PAGE上的标记蛋白测定分子量;其为约110 kDa。通过与凝血酶孵育12小时从蛋白质构建体切割GST，并通过Sephadex G-200色谱法（尺寸排阻）分离hCD 98。hCD 98显示62 kDa的表观分子量，如使用SDS-PAGE基于分子量标记物测定的。CD 98的产量为2 mg/L细胞培养物。
The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 A degrees C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni2+-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 A degrees C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.