Cloning and expression of a murine fascin homolog from mouse brain.
The fascins are a widely distributed family of proteins that organize filamentous actin into bundles. We have cloned, sequenced, and expressed the murine homolog. Fascin is most abundant in brain and is found in other tissues including uterus and spleen. The deduced open reading frame encodes a protein of 493 amino acids with a molecular mass of 54,412 Da. Previous solubility problems with bacterially expressed fascins were overcome by producing the mouse protein as a fusion with Escherichia coli thioredoxin. A method for cleaving the fusion protein and for purifying active recombinant fascin is described. The N-terminal sequence and molecular mass estimated on SDS gels indicate that recombinant fascin is full-length. Two-dimensional gel electrophoresis suggests that recombinant fascin is post-translationally modified in a manner similar to that observed in mouse brain. Recombinant fascin and the fusion protein are recognized by monoclonal anti-fascin antibodies and will bundle rabbit skeletal muscle F-actin in vitro at a stoichiometry of 4.1:1 actin to fascin. Electron cryomicroscopy images show that the reconstituted bundles are highly ordered. However, their fine structure differs from that of echinoid fascin-actin bundles. This structural difference can be attributed to fascin.
Edwards
R A
RA
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Herrera-Sosa
H
H
Otto
J
J
Bryan
J
J
D000107:Acetylation; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
D000199:Actins; D002352:Carrier Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; C020324:fascin