Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase

T. Miyagi, K. Konno, Y. Emori, H. Kawasaki, K. Suzuki, A. Yasui, S. Tsuiki

    Research output: Contribution to journalArticlepeer-review

    111 Citations (Scopus)

    Abstract

    We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.

    Original languageEnglish
    Pages (from-to)26435-26440
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume268
    Issue number35
    Publication statusPublished - 1993

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Fingerprint Dive into the research topics of 'Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase'. Together they form a unique fingerprint.

    Cite this