The expression of recombinant large myelin-associated glycoprotein cytoplasmic domain and the purification of native myelin-associated glycoprotein from rat brain and peripheral nerve.
The myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily existing as two isoforms (L-MAG and S-MAG) that are differentially expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively. The polypeptide chains of the two isoforms differ only by the carboxy terminus of their respective cytoplasmic domains, which most probably determine the isoform-specific functions. Here, we describe the expression of the L-MAG cytoplasmic domain as a GST fusion protein. The recombinant protein was used to raise polyclonal antibodies against the L-MAG-specific carboxy terminus and against the region of the MAG cytoplasmic domain common to both S-MAG and L-MAG. These antibodies, which function in dot blotting, Western blotting, and immunoprecipitation, were used to immunopurify native MAG from both rat brain and peripheral nerves in quantities and purity sufficient for the realization of most biochemical and functional studies. The antibodies and the recombinant and native MAG proteins provide much needed tools for the study of the common and isoform-specific properties and functions of L-MAG and S-MAG.
Heape
A M
AM
Department of Pathology, Oulu University and Central Hospital, Kajaanintie 52D, Oulu, FIN-90220, Finland. Anthony.Heape@oulu.fi
Lehto
V P
VP
Kursula
P
P
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D018992:Myelin-Associated Glycoprotein / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009457:Neuroglia / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012584:Sciatic Nerve / Q000378:metabolism*
D018076:DNA, Complementary; D018992:Myelin-Associated Glycoprotein; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins