Department of Periodontics and Biochemistry, University of Texas Health Science Center at San Antonio, TX 78229-3900, USA.
J Periodontal Res. 2010 Apr;45(2):292-5. doi: 10.1111/j.1600-0765.2009.01241.x. Epub 2009 Nov 9.
Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN.
Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration.
Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly.
The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.
纤连蛋白（FN）是一种重要的细胞黏附分子，广泛用于表征细胞行为。通过明胶-琼脂糖亲和层析从人血浆中纯化的 FN 制剂通常还含有明胶结合的明胶酶，这些明胶酶可能会切割 FN，降低其稳定性并改变其生物学活性。现有的从 FN 中分离明胶酶的方法需要大量资源。因此，我们的目标是设计一种时间和成本效益高的方法来纯化无明胶酶的 FN。
实验测试了 FN 和明胶酶从明胶-琼脂糖上洗脱的洗脱曲线，使用了 DMSO（1-7%）和 4 M 尿素作为洗脱液的浓度范围。随后，我们探索了使用单个亲和柱对明胶酶和 FN 进行顺序洗脱的方法。最后，实验研究了含有或不含有污染明胶酶的纯化 FN 的稳定性，以及 FN 降解对细胞黏附和迁移的影响。
优化实验表明，预洗脱 3% DMSO 可有效地从明胶-琼脂糖上清除明胶酶而不清除 FN，而随后用 4 M 尿素洗脱可释放 FN。DMSO 和尿素的顺序洗脱可产生无明胶酶的 FN，其稳定性比仅用尿素洗脱的 FN 更高。FN 降解不影响人牙龈成纤维细胞的黏附，但显著增加细胞迁移。
本实验设计了一种时间和成本效益高的方法，用于在纯化人血浆 FN 时去除明胶酶。无明胶酶的 FN 制剂具有更高的稳定性，这对于实验可能是必不可少的，因为 FN 片段与完整 FN 相比具有改变的生物学活性。
