Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies

Xinglei Liu, Arne Gennerich

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages69-90
Number of pages22
DOIs
StatePublished - 2024

Publication series

NameMethods in Molecular Biology
Volume2694
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Baculovirus
  • Fluorescence
  • Insect cells
  • KIF5A
  • Kinesin
  • Microtubules
  • Motor proteins
  • Optical tweezers
  • Plasmid
  • Single molecule

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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