Expression and characterization of a single recombinant proteoglycan tandem repeat domain of link protein that binds zinc and hyaluronate.
Expression and characterization of a single recombinant proteoglycan tandem repeat domain of link protein that binds zinc and hyaluronate.
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结合锌和透明质酸盐的连接蛋白的单个重组蛋白聚糖串联重复结构域的表达和表征。
DOI：
10.1006/abbi.1995.1363
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发表时间：
1995
影响因子：
3.9
通讯作者：
Hering,TM
中科院分区：
文献类型：
--
作者：
Varelas,JB;Kollar,J;Huynh,TD;Hering,TM
牛链接蛋白的首个蛋白聚糖串联重复序列（PTR）在pMAL-c载体上被克隆，并在大肠杆菌中与麦芽糖结合蛋白（MBP）融合过表达。融合蛋白可以通过直链淀粉亲和层析从细菌裂解物的可溶性相中分离出来。PTR结构域可以用Xa因子蛋白酶从MBP结构域中分离出来。锌亲和层析表明，牛连接蛋白的锌结合位点中至少有一个包含在第一个PTR结构域内。然后采用锌亲和层析作为MBP/PTR蛋白的最后纯化步骤。MBP/PTR与透明质酸（HA）结合的证据是通过使用十六烷基吡啶氯与HA共沉淀证明的。结合是特异性的，因为MBP/PTR不与硫酸软骨素共沉淀。在ha包被板上的酶联免疫吸附试验中也证实了这种结合。在这个实验中，可以通过添加HA或HA低聚糖来抑制结合。
The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.