TITLE: Purification of NAP1 or GST-NAP1
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes purification of GST-NAP1 and unlabelled NAP1.

[STEPS]
SECTION: Purification of NAP1 or GST-NAP1
1. To purify NAP1 or GST-NAP1, synthesize or clone human NAP1 cDNA in a pGEX-4T1 vector with an N-terminal GST tag followed by a TEV cleavage site (RRID:Addgene_208870).
SECTION: Purification of NAP1 or GST-NAP1
2. For expression of GST-TEV-NAP1 in E. coli, transform the pGEX-4T1 vector encoding GST-TEV-NAP1 into E. coli Rosetta pLySS cells. Grow the cells in 2xTY medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C.
SECTION: Purification of NAP1 or GST-NAP1
3. Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 960 min at 18 °C. 
SECTION: Purification of NAP1 or GST-NAP1
4. Collect the cells by centrifugation and resuspend it in lysis buffer.
Lysis buffer
      Tris-HCl pH 7.4  50 mM    NaCl   300 mM    DTT   1 mM    MgCl2   2 mM    glycerol  5%    β-mercaptoethanol   2 mM     cOmplete EDTA-free protease inhibitors
  (Roche)     DNase (Sigma)  

SECTION: Purification of NAP1 or GST-NAP1
5. Sonicate cell lysates.
SECTION: Purification of NAP1 or GST-NAP1
5.1. Sonicate cell lysates for 30 s. (1/2)
SECTION: Purification of NAP1 or GST-NAP1
5.2. Sonicate cell lysates for 30 s. (1/2)
SECTION: Purification of NAP1 or GST-NAP1
6. Clear the lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification of NAP1 or GST-NAP1
7. Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-TEV-NAP1.
SECTION: Purification of NAP1 or GST-NAP1
8. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of NAP1 or GST-NAP1
9. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash buffer 
      Tris-HCl pH 7.4  50 mM    NaCl  300 mM    glycerol  5%    DTT  1 mM  

High-salt wash buffer 
      Tris-HCl pH 7.4  50 mM    NaCl  700 mM    glycerol  5%    DTT  1 mM  

SECTION: Purification of NAP1 or GST-NAP1
10. Incubate beads 120 min at 4 °C with TEV protease (for unlabeled NAP1) or 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer (for GST-NAP1).
SECTION: Purification of NAP1 or GST-NAP1
11. After the proteins were released from the beads, filter the GST-NAP1 protein through a   syringe filter, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore), or 10 kDa cut-off in case of unlabeled NAP1, and loaded onto a pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva).
SECTION: Purification of NAP1 or GST-NAP1
12. Elute proteins with SEC buffer. 

SEC buffer
      Tris-HCl pH 7.4  25 mM    NaCl  300 mM    DTT  1 mM  

SECTION: Purification of NAP1 or GST-NAP1
13. Analyze fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification of NAP1 or GST-NAP1
14. Pool fractions containing purified NAP1 or GST-NAP1 protein.
SECTION: Purification of NAP1 or GST-NAP1
15. After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store proteins at -80 °C.