Nicotinamide phosphoribosyltransferase purification using SUMO expression system

Trivikram R. Molugu, Radu C. Oita, Udeep Chawla, Sara M. Camp, Michael F. Brown, Joe G.N. Garcia

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via Toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-β-D-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.

Original languageEnglish (US)
Article number113597
JournalAnalytical Biochemistry
Volume598
DOIs
StatePublished - Jun 1 2020

Keywords

  • Biophysical characterization
  • Circular dichroism
  • Dynamic light scattering
  • Ligation-independent cloning
  • NAMPT purification
  • SUMO fusion tag

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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