Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor.
We describe a protocol for purifying hexabrachion from conditioned medium of cell cultures, using gel filtration chromatography on Sephacryl 500, followed by anion-exchange chromatography on a Mono Q column, followed optionally by a second gel filtration or zone sedimentation on glycerol gradients. The protocol has several advantages over previous procedures based on affinity chromatography on monoclonal antibodies. Perhaps foremost, the protein is never exposed to the denaturing solvents that are required for elution from the antibody column. The Mono Q column also separated hexabrachion from a prominent cell adhesion activity that eluted with the hexabrachion on the first gel filtration, and co-sedimented with hexabrachions on glycerol gradients. The cell adhesion fractions showed several bands between 190 and 400 kDa. A single band at 220 kDa stained prominently with a polyclonal antibody against mouse EHS laminin, and a band at 190 kDa stained with a monoclonal antibody against s-laminin. The purification protocol gave hexabrachion at high concentration and with no detectable contamination by fibronectin or laminin. The highest yield of hexabrachion (1-4 mg from 400 ml of conditioned medium) was from human glioblastoma cell cultures, but the same procedure allowed us to purify and characterize the rat hexabrachion. Protein purified from primary cultures of rat embryo fibroblasts showed approximately equal amounts of three subunit sizes: 280, 230, and 220 kDa. These different subunits, presumably derived from alternative RNA splicing, appeared to be segregated into large and small hexabrachions, which could be separated on glycerol gradients.
Department of Periodontics, University of North Carolina School of Dentistry, Chapel Hill 27514.
I
I
Aukhil
C C
CC
Slemp
V A
VA
Lightner
K
K
Nishimura
G
G
Briscoe
H P
HP
Erickson
D001253:Astrocytes / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000302:isolation & purification*; D015816:Cell Adhesion Molecules, Neuronal / Q000302:isolation & purification*; D002850:Chromatography, Gel / Q000379:methods*; D003470:Culture Media / Q000032:analysis*; D005910:Glioma; D006801:Humans; D019063:Tenascin
D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D003470:Culture Media; D019063:Tenascin