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Molecular cloning, tissue expression, and partial characterization of the major fish CNS myelin protein 36k

A full‐length cDNA clone encoding the major structural protein of trout CNS myelin 36K was isolated and sequenced. The deduced amino acid sequence did not reveal a putative transmembrane domain and exhibited no structural homology with any of the known myelin proteins. 36K instead shared characteristic structural elements with enzymes of the short‐chain dehydrogenase family. The highest similarity in the database (60%), however, was obtained with a human protein of unknown function. By Northern blotting, a single mRNA species of about 2 kb was identified, which was expressed in brain tissue but not in liver. By in situ hybridization, a selective labeling of myelinating glial cells in the trout CNS but not in the PNS was revealed. The developmental appearance of the 36K transcript closely coincided with a period of active myelin deposition in most regions of the trout brain. As a first step in elucidating the structural and biochemical role of 36K for myelin formation and maintenance, we have overexpressed it in Escherichia coli as a soluble His‐tag fusion protein and purified it in high yield by Ni+‐chelated affinity chromatography. By SDS‐PAGE, a single band of the expected molecular size was revealed, which heavily cross‐reacted with polyclonal antibodies generated against the native protein. The results of circular dichroism spectroscopy are compatible with a βαβ‐barrel structure (Rossman fold), confirming the results of computer‐assisted secondary structure predictions. © 2003 Wiley‐Liss, Inc.