Purification of MBP-NAP1
Elias Adriaenssens
Abstract
This protocol describes purification of MBP-TEV-NAP1 protein.
Attachments
Steps
Purification of MBP-NAP1
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).
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.
Once the cells reached an OD600 of 0.8, induce protein expression with 50micromolar (µM) IPTG for 16h 0m 0s at 18°C.
Collect the cells by centrifugation and resuspend them in lysis buffer.
Lysis buffer
| A | B |
|---|---|
| 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) |
Sonicate cell lysates and then clear by centrifugation.
Sonicate cell lysates for 0h 0m 30s. (1/2)
Sonicate cell lysates for 0h 0m 30s. (2/2)
Then, centrifugation at 18000rpm,4°C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
Collect and incubate the supernatant with pre-equilibrated Amylose beads (Biolabs) for 2h 0m 0s at 4°C with gentle shaking to bind MBP-TEV-NAP1.
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.
Wash buffer
| A | B |
|---|---|
| Tris-HCl pH 7.4 | 50 mM |
| NaCl | 300 mM |
| glycerol | 5% |
| DTT | 1 mM |
High-salt wash buffer
| A | B |
|---|---|
| Tris-HCl pH 7.4 | 50 mM |
| NaCl | 700 mM |
| glycerol | 5% |
| DTT | 1 mM |
Incubate the beads 2h 0m 0s at 4°C with 250millimolar (mM) D-maltose (Santa Cruz) dissolved in wash buffer.
After the proteins are released from the beads, filter the MBP-TEV-NAP1 protein through a 0.45 µm 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).
Elute the proteins with SEC buffer.
SEC buffer
| A | B |
|---|---|
| Tris-HCl pH 7.4 | 25 mM |
| NaCl | 300 mM |
| DTT | 1 mM |
Analyze the fractions by SDS-PAGE and Coomassie staining.
Pool the fractions containing purified MBP-TEV-NAP1 protein.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at -80°C.
