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Overexpression and purification of human myosins from transiently and stably transfected suspension adapted HEK293SF-3F6 cells
Technion - Israel Institute of Technology, Israel.
Technion - Israel Institute of Technology, Israel.
Technion - Israel Institute of Technology, Israel.
Technion - Israel Institute of Technology, Israel.
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2018 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 558, no 1, p. 19-27Article in journal (Refereed) Published
Abstract [en]

The myosin family of motor proteins is an attractive target of therapeutic small-molecule protein inhibitors and modulators. Milligrams of protein quantities are required to conduct proper biophysical and biochemical studies to understand myosin functions. Myosin protein expression and purification represent a critical starting point towards this goal. Established utilization of Dictyostelium discoideum, Drosophila melanogaster, insect and mouse cells for myosin expression and purification is limited, cost, labor and time inefficient particularly for (full-length) human myosins. Here we are presenting detailed protocols for production of several difficult-to-purify recombinant human myosins in efficient quantities up to 1 mg of protein per liter of cell culture. This is the first time that myosins have been purified in large scales from suspension adapted transiently and stably expressing human cells. The method is also useful for expressing other human proteins in quantities sufficient to perform extensive biochemical and biophysical characterization.

Place, publisher, year, edition, pages
Elsevier, 2018. Vol. 558, no 1, p. 19-27
Keywords [en]
Recombinant myosins, Human cell expression system, HEK293SF-3F6, Suspension cell culture, Gene expression, Polyethyleneimine
National Category
Biochemistry and Molecular Biology Biophysics Cell Biology
Research subject
Chemistry, Biotechnology; Chemistry, Biochemistry; Natural Science, Cell and Organism Biology
Identifiers
URN: urn:nbn:se:lnu:diva-81832DOI: 10.1016/j.ab.2018.07.026OAI: oai:DiVA.org:lnu-81832DiVA, id: diva2:1304020
Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-05-03Bibliographically approved

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Ušaj, Marko

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CiteExportLink to record
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Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf