Purified recombinant lentiviral Vpx proteins maintain their SAMHD1 degradation efficiency in resting CD4 + T cells.

Autor: Nair R; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany., Pignot Y; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany., Salinas-Illarena A; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany., Bärreiter VA; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany., Wratil PR; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany; German Center for Infection Research (DZIF), Partner Site Munich, 80802 Munich, Bavaria, Germany., Keppler OT; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany., Wichmann C; Division of Transfusion Medicine, Cell Therapeutics and Haemostaseology, University Hospital, LMU Munich, Munich, Germany., Baldauf HM; Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, Faculty of Medicine, LMU München, Munich, Germany. Electronic address: baldauf@mvp.lmu.de.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2023 Jun 01; Vol. 670, pp. 115153. Date of Electronic Publication: 2023 Apr 08.
DOI: 10.1016/j.ab.2023.115153
Abstrakt: Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4 + T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4 + T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.
Competing Interests: Declaration of competing interest O.T.K. and H.-M.B. are listed as inventors of patent no. WO2017076880A1. All other authors declare no competing interests.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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