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dc.contributor.authorRaducanu, Vlad-Stefan
dc.contributor.authorTehseen, Muhammad
dc.contributor.authorShirbini, Afnan
dc.contributor.authorRaducanu, Daniela-Violeta
dc.contributor.authorHamdan, Samir
dc.date.accessioned2020-03-19T11:37:44Z
dc.date.available2020-03-19T11:37:44Z
dc.date.issued2020-03-17
dc.date.submitted2019-11-28
dc.identifier.citationRaducanu, V.-S., Tehseen, M., Shirbini, A., Raducanu, D.-V., & Hamdan, S. M. (2020). Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions. Journal of Chromatography A, 461051. doi:10.1016/j.chroma.2020.461051
dc.identifier.doi10.1016/j.chroma.2020.461051
dc.identifier.urihttp://hdl.handle.net/10754/662205
dc.description.abstractThe strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.
dc.description.sponsorshipThe authors would like to thank Dr. Mohamed A. Sobhy for critical reading of the manuscript and for his valuable feedback.
dc.description.sponsorshipThis work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award [CRG3 to S.M.H.].
dc.publisherElsevier BV
dc.relation.urlhttps://linkinghub.elsevier.com/retrieve/pii/S0021967320302636
dc.rightsNOTICE: this is the author’s version of a work that was accepted for publication in Journal of Chromatography A. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Chromatography A, [[Volume], [Issue], (2020-03-17)] DOI: 10.1016/j.chroma.2020.461051 . © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.titleTwo chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions
dc.typeArticle
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.contributor.departmentBioscience Program
dc.contributor.departmentLaboratory of DNA Replication and Recombination
dc.identifier.journalJournal of Chromatography A
dc.rights.embargodate2022-03-17
dc.eprint.versionPost-print
kaust.personRaducanu, Vlad-Stefan
kaust.personTehseen, Muhammad
kaust.personShirbini, Afnan
kaust.personRaducanu, Daniela-Violeta
kaust.personHamdan, Samir
dc.date.accepted2020-03-15
refterms.dateFOA2020-03-19T11:39:08Z
dc.date.published-online2020-03-17
dc.date.published-print2020-06


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