TITLE: Purification of MBP-NAP1
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes purification of MBP-TEV-NAP1 protein.

[STEPS]
SECTION: Purification of MBP-NAP1
1. To purify MBP-NAP1, gene-synthesize human NAP1 cDNA (by Genscript) and subclone into a pGEX-4T1 vector with an N-terminal MBP-tag. Follow it by a TEV cleavage site before wild-type NAP1 (RRID:Addgene_208871), NAP1 delta-NDP52 (S37K/A44E) (RRID:Addgene_208872), or NAP1 delta-TBK1 (L226Q/L233Q) (RRID:Addgene_208873).
SECTION: Purification of MBP-NAP1
2. For expression of MBP-TEV-NAP1 in E. coli, transfer the pGEX-4T1 vector encoding MBP-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 MBP-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 MBP-NAP1
4. Collect the cells by centrifugation and resuspend them 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 MBP-NAP1
5. Sonicate cell lysates and then clear by centrifugation.
SECTION: Purification of MBP-NAP1
5.1. Sonicate cell lysates for 30 s. (1/2)

SECTION: Purification of MBP-NAP1
5.2. Sonicate cell lysates for 30 s. (2/2)
SECTION: Purification of MBP-NAP1
5.3. Then, centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
SECTION: Purification of MBP-NAP1
6. Collect and incubate the supernatant with pre-equilibrated Amylose beads (Biolabs) for 120 min at 4 °C with gentle shaking to bind MBP-TEV-NAP1.
SECTION: Purification of MBP-NAP1
7. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of MBP-NAP1
8. 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 MBP-NAP1
9. Incubate the beads 120 min at 4 °C with 250 millimolar (mM) D-maltose (Santa Cruz) dissolved in wash buffer.
SECTION: Purification of MBP-NAP1
10. After the proteins are released from the beads, filter the MBP-TEV-NAP1 protein through a   syringe filter, concentrate using a 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva). 
SECTION: Purification of MBP-NAP1
11. Elute the proteins with SEC buffer. 
SEC buffer
      Tris-HCl pH 7.4  25 mM    NaCl  300 mM    DTT  1 mM  

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