Extraction and purification of decorin from corneal stroma retain structure and biological activity
Extraction and purification of decorin from corneal stroma retain structure and biological activity
复制标题
DOI：
10.1016/s1046-5928(02)00025-6
复制
发表时间：
2002-08-01
影响因子：
1.6
通讯作者：
Trinkaus-Randall, V
中科院分区：
文献类型：
--
作者：
Brown, CT;Lin, P;Trinkaus-Randall, V
我们开发了一种从组织中纯化核心蛋白聚糖核心蛋白的方法，目的是保留其天然结构和生物学功能。目前，大多数程序依赖于变性试剂的使用，可能会改变生物活性。核心蛋白聚糖从角膜基质中纯化，而不使用去污剂或离液试剂。用软骨素酶ABC处理在Q-Sepharose上使用阴离子交换色谱分离的蛋白聚糖。通过第二次Q-Sepharose层析与肝素-Sepharose和伴刀豆球蛋白A-Sepharose上的亲和层析分离核心蛋白聚糖。SDS-PAGE显示纯度为98.4%的44 kDa蛋白被鉴定为核心蛋白聚糖，产量为35 mg/100个牛角膜。通过NanoESI和MALDI qTOF确认鉴别。在提取和柱缓冲液中加入20%丙二醇的新方法使蛋白聚糖的回收率与用洗涤剂和尿素观察到的回收率相当。纯化的核心蛋白聚糖确实改变了I型胶原的原纤维形成速率，并抑制了胶原原纤维的侧向融合。它还与[I-125] TGF-β 1结合，表观Kd为40 nM。核心蛋白聚糖的圆二色性光谱显示与从重组核心蛋白聚糖获得的那些一致的α-螺旋和β-折叠片的光谱。尿素诱导的去折叠是协同的和可逆的，而热变性导致不可逆的去折叠。天然核心蛋白聚糖可以从组织中大量纯化并用于生物物理学。生物化学和生物测定。(C)2002 Elsevier Science（美国）。All rights reserved.
We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44 kDa protein identified as decorin with a yield of 35 mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [I-125]TGF-beta1 with an apparent K-d of 40 nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical. biochemical, and biological assays. (C) 2002 Elsevier Science (USA). All rights reserved.