Expression and purification of large nebulin fragments and their interaction with actin

Biophysical Journal
J Q ZhangR Horowits

Abstract

cDNA clones encoding mouse skeletal muscle nebulin were expressed in Escherichia coli as thioredoxin fusion proteins and purified in the presence of 6 M urea. These fragments, called 7a and 8c, contain 28 and 19 of the weakly repeating approximately 35-residue nebulin modules, respectively. The nebulin fragments are soluble at extremely high pH, but aggregate when dialyzed to neutral pH, as assayed by centrifugation at 16,000 x g. However, when mixed with varying amounts of G-actin at pH 12 and then dialyzed to neutral pH, the nebulin fragments are solubilized in a concentration-dependent manner, remaining in the supernatant along with the monomeric actin. These results show that interaction with G-actin allows the separation of insoluble nebulin aggregates from soluble actin-nebulin complexes by centrifugation. We used this property to assay the incorporation of nebulin fragments into preformed actin filaments. Varying amounts of aggregated nebulin were mixed with a constant amount of F-actin at pH 7.0. The nebulin aggregates were pelleted by centrifugation at 5200 x g, whereas the actin filaments, including incorporated nebulin fragments, remained in the supernatant. Using this assay, we found that nebulin fragments 7a and 8c...Continue Reading

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Citations

Mar 8, 2000·Cell Motility and the Cytoskeleton·A H HerreraR Horowits
Jan 19, 1999·Annual Review of Cell and Developmental Biology·R Littlefield, V M Fowler
Jul 21, 2010·Journal of Molecular Biology·Asa K BjörklundArne Elofsson
Jul 11, 2008·Cell Motility and the Cytoskeleton·Shajia Lu, Robert Horowits
May 15, 2010·Journal of Biomedicine & Biotechnology·Ryo ChitoseSumiko Kimura
Mar 8, 2002·Current Biology : CB·Natalya LukoyanovaEdward H Egelman
Aug 10, 2000·The Journal of Cell Biology·K OjimaH Holtzer
Oct 4, 2000·The Journal of Biological Chemistry·A S McElhinnyC C Gregorio
Nov 26, 2002·The Journal of Biological Chemistry·Ozgur OgutJian-Ping Jin

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