sepharose and cesium-chloride

Here we describe a single-step affinity column for purification of vectors based on adeno-associated virus type 5 (AAV5). A sialic-acid-rich protein called mucin was covalently attached to Sepharose and was found to bind AAV5 vectors. Elution with high salt efficiently recovered highly active vectors of greater purity than what is achieved with CsCl(2) sedimentation.

Topics: Animals; Cell Line; Cesium; Chlorides; Chromatography, Affinity; Dependovirus; Genetic Vectors; Humans; Male; Mice; Mice, Inbred C57BL; Mucins; Muscle, Skeletal; Salts; Sepharose; Ultracentrifugation

A previous lectin binding study demonstrated the presence of high molecular-mass mucin-like glycoproteins (HMGP) on the surface of hamster tracheal surface epithelial (HTSE) secretory cells (Proc. Natl. Acad. Sci. USA 1987;84:9304). In the present study, we intended to isolate and characterize these HMGP from the plasma membrane of the primary HTSE cells and then to determine whether or not these membrane HMGP are Muc-1 mucins, a type of mucins originally discovered on the surface of some carcinomas. A subcellular fraction enriched with the plasma membrane was obtained using a sucrose density gradient centrifugation. This fraction contained high molecular-mass glycoconjugates which were excluded from Sepharose CL-4B gel. Biochemical characterization of these glycoconjugates revealed the following characteristics: (1) susceptibility to both pronase and mild alkaline treatments, but totally resistant to proteoglycan-digesting enzymes; (2) partitioning in the detergent phase of Triton X-114 and resistance to digestion by phosphatidylinositol phospholipase C or D; (3) a buoyant density of 1.5 g/ml based on CsCl density gradient centrifugation; (4) polydispersity in terms of both size and charge density; and (5) lack of immunoreactivity with an anti-Muc-1 mucin antibody. We conclude that the plasma membrane of HTSE cells at confluence contains HMGP, which seem to be the integral membrane proteins but different from Muc-1 mucins, and that these membrane HMGP appear to share some similarities with secreted mucins in terms of size and charge.

Topics: Animals; Antibodies; Cell Fractionation; Cell Membrane; Centrifugation; Cesium; Chlorides; Chromatography; Cricetinae; Detergents; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Ethanolamines; Glycoproteins; Male; Mesocricetus; Molecular Weight; Mucins; N-Acetylneuraminic Acid; Octoxynol; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase D; Polyethylene Glycols; Precipitin Tests; Rabbits; Sepharose; Trachea; Tritium; Type C Phospholipases

Topics: Cesium; Chlorides; Dialysis; Gels; Macromolecular Substances; Polysaccharides; Salts; Sepharose

Bovine oestrus cervical mucin, isolated by gel filtration on Sepharose 6B, was found to be homogeneous (i) in electrophoresis on cellulose acetate at pH 8.6 after release of sialic acid and in acrylamide-agarose gel at pH 8.7 in the presence of sodium dodecyl sulphate, where not a single compact zone was found after staining for proteins, (ii) by analytical ultracentrifugation and equilibrium centrifugation in a CsCl density gradient; as well as (iii) by immunoelectrophoresis, which revealed two kinds of antibodies against mucin, reacting, respectively, with the primary structure and with determinants depending on the integrity of the glycoprotein structure. Alanine was identified as the N-terminal amino acid of the peptide core, which appeared to consist of a repetition of a 32-35 residue sequence, viz. (Thr)8, (Glu, Pro, Ala, Val)3, (Asp, Leu)2 and (Arg, Cys, Ileu)1. No integral numbers could be assigned to (Ser)3.4, (Gly)2.3 and (Phe)0.6 which were also present.

Topics: Acrylamides; Amino Acids; Animals; Cattle; Cervix Uteri; Cesium; Chlorides; Cross-Linking Reagents; Estrus; Female; Hydrogen-Ion Concentration; Mucins; Peptide Hydrolases; Protein Structure, Tertiary; Sepharose; Ultracentrifugation