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Nicotinamide phosphoribosyltransferase purification using SUMO expression system.

Trivikram R Molugu1, Radu C Oita2, Udeep Chawla1

  • 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, USA.

Analytical Biochemistry
|January 27, 2020
PubMed
Summary

Researchers successfully expressed and purified Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme crucial for NAD+ synthesis and inflammation. This purified NAMPT protein, in monomeric form, facilitates further structural and functional studies.

Keywords:
Biophysical characterizationCircular dichroismDynamic light scatteringLigation-independent cloningNAMPT purificationSUMO fusion tag

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in NAD+ biosynthesis.
  • Secreted NAMPT acts as a cytokine, activating inflammatory pathways.
  • NAMPT's role as a damage-associated molecular pattern (DAMP) protein links it to various disorders via TLR4.
  • Limited availability of purified NAMPT hinders detailed functional and structural studies.

Purpose of the Study:

  • To develop a method for expressing and purifying functional Nicotinamide phosphoribosyltransferase (NAMPT) protein.
  • To characterize the biophysical properties and oligomeric state of purified NAMPT.
  • To enable further structural and functional investigations of NAMPT.

Main Methods:

  • Expression of NAMPT in E. coli using the pET-SUMO vector with a hexa-His tag.
  • Purification via immobilized-metal affinity chromatography after SUMO and His tag cleavage.
  • Biophysical characterization using circular dichroism (CD) and dynamic light scattering (DLS).
  • Solubilization of NAMPT in n-dodecyl-β-d-maltopyranoside (DDM) detergent.

Main Results:

  • Successful expression and purification of functional NAMPT protein with a yield of approximately 4 mg/L.
  • Circular dichroism confirmed secondary structural elements, and DLS indicated the presence of oligomeric units.
  • The NAMPT-SUMO fusion protein was predominantly dimeric.
  • NAMPT was successfully solubilized in DDM detergent in a monomeric form.

Conclusions:

  • A robust method for producing purified, enzymatically active NAMPT protein has been established.
  • The availability of monomeric NAMPT in detergent enhances opportunities for detailed structural and functional analyses.
  • This work provides a foundation for understanding NAMPT's role in inflammatory processes and related diseases.