Purification of NAP1 or GST-NAP1
Elias Adriaenssens
Abstract
This protocol describes purification of GST-NAP1 and unlabelled NAP1.
Attachments
840-2174.pdf
Steps
Purification of NAP1 or GST-NAP1
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).
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.
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 it 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.
Sonicate cell lysates for 0h 0m 30s. (1/2)
Sonicate cell lysates for 0h 0m 30s. (1/2)
Clear the lysates by 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 Glutathione Sepharose 4B beads (GE Healthcare) for 2h 0m 0s at 4°C with gentle shaking to bind GST-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 beads 2h 0m 0s at 4°C with TEV protease (for unlabeled NAP1) or 4mL of 50millimolar (mM) reduced glutathione dissolved in wash buffer (for GST-NAP1).
After the proteins were released from the beads, filter the GST-NAP1 protein through a 0.45 µm 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).
Elute proteins with SEC buffer.
SEC buffer
| A | B |
|---|---|
| Tris-HCl pH 7.4 | 25 mM |
| NaCl | 300 mM |
| DTT | 1 mM |
Analyze fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified NAP1 or GST-NAP1 protein.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store proteins at -80°C.
