A gelsolin-related protein from lobster muscle: cloning, sequence analysis and expression.
The tail muscle of the lobster Homarus americanus contains an actin-binding protein with an apparent molecular mass of 105 kDa determined by SDS/PAGE and gelsolin-like properties. We isolated this protein and peptide sequences were obtained after limited proteolysis with chymotrypsin. A tail-muscle-specific cDNA library was constructed in a lambda expression vector and a full-length clone was obtained by screening with a polyclonal anti-(crustacean gelsolin) antibody. The cDNA insert of approx. 3.2 kb length was sequenced. The cDNA contained an open reading frame of 2.265 kb, and the deduced amino acid sequence of 754 residues (83,469 Da) identified the protein as a cytoplasmic member of the gelsolin/villin protein family. Comparison of the lobster gelsolin amino acid sequence with other members of this protein family revealed the characteristic 6-fold repeated segmental structure as well as the three conserved sequence motifs typical of each segment [Way and Weeds (1988) J. Mol. Biol. 203, 1127-1133]. Strong homologies were found with Drosophila gelsolin, human gelsolin, villin core, Dictyostelium severin and Physarum fragmin. In addition, the gelsolin-like protein from lobster muscle revealed motifs that were clearly similar to the actin-bundling region of human villin headpiece although it did not itself contain a distinct headpiece domain. The recombinant lobster gelsolin-like protein, expressed in Escherichia coli as a fusion protein, was purified from inclusion bodies and renatured as a functional protein. There were no significant differences in the biological activity tested between the recombinant and the native protein isolated from lobster muscle.
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Biochemical Cell Biology Group, University of Bielefeld, Federal Republic of Germany.
D'Haese
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Hinssen
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D018260:Gelsolin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D008121:Nephropidae; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D018076:DNA, Complementary; D018260:Gelsolin; D011993:Recombinant Fusion Proteins; D002918:Chymotrypsin