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Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations

Publication Type : Journal Article

Thematic Areas : Biotech

Publisher : Wiley Online Library

Source : Journal of periodontal research, Wiley Online Library, Volume 45, Number 2, p.292–295 (2010)

Campus : Amritapuri

School : School of Biotechnology

Center : Cancer Biology

Department : biotechnology

Year : 2010

Abstract : Background and Objective: 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. Material and Methods: 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. Results: 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. Conclusion: 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. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard.

Cite this Research Publication :
Dr. Sanjay Pal, Chen, Z., Xu, X., Mikhailova, M., and Steffensen, B., “Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations”, Journal of periodontal research, vol. 45, pp. 292–295, 2010

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