Large Scale Affinity Chromatography (NEB #E8201)
This protocol is for purification of a fusion protein from a 1-liter culture.
MBP Column Binding Buffer:
20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT (optional), pH 7.5.
- Inoculate 1-liter rich broth supplemented with 0.2% glucose & 100 μg/ml ampicillin with 10 ml of an overnight culture of cells containing the fusion plasmid.
Glucose is necessary in the growth medium to repress the maltose genes on the chromosome of the E. coli host, one of which is an amylase which can degrade the amylose on the affinity resin.
- Grow to 2 x 108 cells/ml (OD600 ~0.5). Add IPTG to a final concentration of 0.3 mM, e.g., 72 mg or 3 ml of a 0.1 M stock in H2O. Incubate the cells at 37°C for 2 hours.
The length of time and the temperature to use during expression depends on several factors and variations can be tested to find optimum conditions.
- Harvest the cells by centrifugation at 4000 x g for 20 minutes and discard the supernatant. Resuspend the cells in 25 ml Column Buffer.
It is best to harvest cells quickly and keep them chilled. 25 ml of Column Buffer is based on the expectation of about 4-5 grams cells/liter, i.e., 5 ml for every gram of cells (wet weight). The EDTA in the lysis buffer is to help inhibit proteases that have a Ca2+ cofactor. Addition of PMSF or other protease inhibitors may help in some cases. DTT or β–mercaptoethanol can be included to prevent oxidative damage to the fusion protein and interchain disulfide bond formation upon lysis.
- Freeze resuspended cells overnight at –20°C.
This is a good place to stop in this protocol. The frozen cells can be stored for a week or more at –20°C, depending on the particular fusion.
- Thaw the resuspended cells. Do not use lysozyme during lysis, as it can break down the amylose resin. Place metal cup containing the resuspended cells and stir bar in an ice-water bath. Stir cells slowly and sonicate in short pulses of 15 seconds or less. Monitor the release of protein using the Bradford assay, adding 5 μl of the sonicate to 1 ml Bradford reagent and mixing. Continue sonication until the released protein reaches a maximum (~2 minutes sonication time).
- Centrifuge the lysate at 20,000 x g for 20 minutes. Save the supernatant (crude extract). Dilute the crude extract 1:5 with Column Buffer.
This is another good place to stop the protocol. The crude extract can be stored for a week or more at –20°C, depending on the particular fusion.
- Pour the amylose resin in a 2.5 x 10 cm column. Wash the column with 5 column volumes of Column Buffer.
The amount of resin required depends on the amount of fusion protein produced. The resin binds 4 mg/ml bed volume, thus a 25 ml column is sufficient for a yield of up to 100 mg fusion protein/liter culture.
- Load the diluted crude extract at a flow rate of no more than [50 x (diameter of column in cm)2] ml/hour. This is a maximum of 5 ml/minute for a 2.5 cm column. Slower flow rates may increase MBP-fusion binding to the amylose resin; however, the crude extract should not be incubated with the amylose resin overnight, because native E. coli amylases can break down the resin and decrease its binding capacity.
- Wash with 12 column volumes of Column Buffer at a rate of no more than [100 x (diameter of column in cm)2] ml/hour. This is a maximum of 10 ml/minute for a 2.5 cm column.
The column can be washed overnight if it has a safety loop to prevent it from running dry. In this case, it is better to restart the column with elution buffer (Step 10), rather than continuing the wash. Avoid loading the column overnight.
- Elute the fusion protein with Column Buffer + 10 mM maltose. Collect 10 to 20 3 ml fractions (fraction size = 1/5th column volume). The fusion protein usually starts to elute within the first 5 fractions and should be easily detected by UV absorbance at 280 nm or the Bradford protein assay.
- Pool the protein-containing fractions. If necessary, concentrate to ~1 mg/ml in a Vivaspin Centrifugal Concentrator (Sartorius), or the equivalent.