A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium.
Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans or other products of smooth muscle cell metabolism.
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000032:analysis; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D010779:Photofluorography; D011347:Procollagen / Q000302:isolation & purification*; D011487:Protein Conformation; D011817:Rabbits
D003470:Culture Media; D005353:Fibronectins; D011347:Procollagen