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

Anal Biochem. 2023 Jun 1:670:115153. doi: 10.1016/j.ab.2023.115153. Epub 2023 Apr 8.

Abstract

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.

Keywords: CD4(+) T cells; Functionality; HEK293T; IMAC; Nucleofection; Protein purification; Recombinant protein; Vpx.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD4-Positive T-Lymphocytes
  • HEK293 Cells
  • Humans
  • Monomeric GTP-Binding Proteins* / chemistry
  • Monomeric GTP-Binding Proteins* / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • SAM Domain and HD Domain-Containing Protein 1 / genetics
  • SAM Domain and HD Domain-Containing Protein 1 / metabolism
  • T-Lymphocytes*
  • Viral Regulatory and Accessory Proteins / genetics
  • Viral Regulatory and Accessory Proteins / metabolism

Substances

  • SAM Domain and HD Domain-Containing Protein 1
  • Recombinant Proteins
  • Monomeric GTP-Binding Proteins
  • VPX protein, Human immunodeficiency virus 2
  • Viral Regulatory and Accessory Proteins
  • SAMHD1 protein, human