Inducible expression in human cells, purification, and in vitro assays for the mitochondrial DNA helicase Twinkle.
Mitochondrial DNA (mtDNA) maintenance can be and has been studied in a wide variety of organisms, some more tractable than others. We use human and mouse cell culture models to study proteins and mechanisms of mtDNA replication. Recent advances in cell culture systems allow us to streamline the analysis of replication proteins both in vivo in cell culture and in vitro following protein purification. One such system, the inducible 293 Flp-In(TM) TREx(TM) system, will be described here in detail with the emphasis on the mitochondrial DNA helicase Twinkle, in particular its mitochondrial purification following over-expression, and basic activity and multimerization assays.
Goffart
Steffi
S
Institute of Medical Technology, University of Tampere, Tampere, Finland.
Spelbrink
Hans
H
D001681:Biological Assay; D002478:Cells, Cultured; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D024101:Mitochondrial Proteins; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D004265:DNA Helicases; C431576:TWNK protein, human