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Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications

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Abstract

The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.

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Abbreviations

bp:

Base pairs

C:

Carboxy

Cy5:

Cyanine 5

EDTA:

Ethylenediaminetetraacetic acid

EMSAs:

Electrophoretic mobility shift assays

FBS:

Fetal bovine serum

FRT:

Flp recognition target

kDa:

Kilo Dalton

PAGE:

Polyacrylamide gel electrophoresis

P/S:

Penicillin–streptomycin

SDS:

Sodium dodecyl sulfate

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Acknowledgements

We thank Juan Yuan for help with spectrometry, and Susanne Stubbe and Golshah Ayoubi for excellent technical assistance. We wish to thank the Danish Neuroscience Centre, Aarhus, Denmark for use of cell culturing facilities. This study was supported by a Lundbeck Foundation Fellowship and the Sapere Aude Program of the Danish Council for Independent Research to MMG. ISJ was supported by the Graduate School of Health, Aarhus University.

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Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark

    Ida S. Jensen, Ken Inui, Srdja Drakulic, Sakthidasan Jayaprakash & Monika M. Golas

  2. Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark

    Bjoern Sander

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  1. Ida S. Jensen
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  2. Ken Inui
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Correspondence to Monika M. Golas.

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Jensen, I.S., Inui, K., Drakulic, S. et al. Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications. Protein J 36, 332–342 (2017). https://doi.org/10.1007/s10930-017-9724-z

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