Affinity chromatography methods can virtually purify maltose-binding fusion proteins into single components. Maltose-binding protein self (MBP) is a periplasmic protein encoded by the malE gene of Escherichia coli and is a member of the bacterial maltose transport system.MBP binds micromolar levels of maltose and maltodextrin, and can therefore be purified with agarose cross-linked with maltose. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experimental purification of maltose-binding fusion proteins by affinity chromatography with straight-chain starch resin
Materials and Instruments
E. coli cells expressing BMP fusion proteins Move makings For more product details, please visit Aladdin Scientific website.
Cell Lysis Buffer Cell Wash Buffer Column Elution Buffer Column Wash Buffer MgCl2 PMSF SDS Gel Spiking Buffer Tris-Cl DNase Lysozyme RNase Thrombin, Enterokinase, or Xa Factor Solution SDS-Polyacrylamide Gel
Beckman Ti60 turn-table or equivalent Sorvall GSA turn-table or equivalent Sorvall SS-34 turn-table or equivalent Straight-chain starch agarose Boiling water bath Ultrasound instrumentation -Substantial Nalgene filters
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
Cell Lysis Buffer (~150 ml)
30 mmol/LTris-Cl (pH 7.l)
0.1 mmol/LEDTA
20% (m/V) sucrose
Cell wash buffer (~250 ml)
10 mmol/L Tris-Cl (pH 7.1)
30 mmol/LNaCl
Column elution buffer (~100 ml)
10 mmol/LTris-Cl (pH 7.5)
10 mmol/L maltose
Column wash buffer (~250 ml)
10 mmol/L Tris-Cl (pH 7.1)
1 mol/L NaCl
MgCl2 (0.1 mmol/L,~250 ml)
PMSF(100mmol/L)
17.4 mg PMSF dissolved in 1 ml isopropanol, stored at -20°C.
PMSF is readily inactivated in aqueous solutions, with the rate of inactivation increasing with the pH of the aqueous solution and faster at 25°C than at 4°C. The half-life of a 20 mmol/L aqueous solution of PMSF at pH 8.0 is about 35 min (James 1978), and this short half-life means that alkaline (pH > 8.6) aqueous PMSF solutions can be safely discarded after a few hours of storage at room temperature. This short half-life means that basic (pH>8.6) aqueous PMSF solutions can be safely discarded after several hours of storage at room temperature.
Alternatives to PMSF can be purchased from Boehringer Mannheim (4-[2-aminoethyl]-benzenesulfonylfluoride, hydrochloride; Pefabloc SC), an irreversible serine protease inhibitor that can be used at the same concentration as PMSF but is non-toxic and stable in aqueous solution.
1XSDS Gel Spiking Buffer
DTT-free 1xSDS gel spiking buffer is stored at room temperature, 1mol/LDTT storage solution is added to the above buffer as it is used.
Tris-Cl (10 mmol/L, pH 7.1)
Enzyme and buffer
DNase(5 mg/ml)
Lysozyme
RNase(5 mg/ml)
Thrombin, enterokinase, or Factor Xa solution
Refer to manufacturer's manual for preparation and storage of solutions.
Gel
SDS-Polyacrylamide Gel (10%)
Refer to Appendix 8 for preparation of SDS-polyacrylamide gels for protein isolation.
Centrifuge and rotor head
Beckman Ti60 turntable or equivalent
Sorvall GSA turntable or equivalent
Sorvall SS-34 Turn Head or equivalent
Special equipment
Gibberellic acid agarose
can be synthesized by the method of Kdlermann and Ferenci (1982) or can be purchased from New England Bioland Public SJ (1 to 4 mg MBP per ml of column bed volume).
Boiling water bath
Ultrasonic apparatus
See Appendix 8.
-Secondary Nalgene Filters (0.45um cellulose nitrate membrane)
Carriers and Bacterial Strains
E. coli cells expressing BMP fusion proteins (cell precipitates prepared in step 17 of Scheme 1 or 2).
Methods
Preparation of straight chain starch agarose columns
1. 3 cmX6 cm columns of straight-chain starch agarose suspended in 10 mmol/L Tris-Cl (pH 7.1) at 4°C were packed.
Preparation of cell extracts
2. Suspend the cell precipitate in ice-cold Cell Wash Buffer equal to 1/10 of the volume of the original culture (usually 50 ml), centrifuge at 5000 g at 4°C (5500 r/min Sovall GSA head) for 15 min to collect the cell precipitate, and then suspend it in ice-cold Fine Holding Wash Buffer equal to 1/10 of the volume of the original culture (usually 50 ml).
3. Preparation of cell lysates
If the vector used does not carry the MBP signaling peptide sequence (pMAL-c2), the cell lysate should be prepared.
a. Ultrasonically break the cells at 30W and keep the cells at a low temperature (0°C).
b. To clarify the solution, add PMSF to a final concentration of 1 mmol/L, and centrifuge at 87,000 g (35,000r/min Beckman Ti60 rotor head) for 30 min at 4°C.
Continue with Step 4, straight chain starch agarose chromatography to purify the fusion protein from the supernatant.
If the vector used carries the MBP peptide sequence (pMAL-p2), the fusion protein is purified from the supernatant by straight-chain starch agarose chromatography.
a. Collect cells by centrifugation at 5000 g (5500 r/min Sorvall GSA turntable) for 15 min at 4°C and suspend the precipitate in ice-cold Cell Lysis Buffer equivalent to 1/20 volume of the original culture (typically 25 ml).
b. Add PMSF to a final concentration of 1 mmol/L, stir at room temperature for 15 min, and centrifuge at 17200 g (12000r/min Sorvall SS-34 head) for 10 min at 4°C to collect cells.
c. Spin the cell sediment, add ice-cold 0.1 mmol/L MgCl2 solution (lOOml per liter of culture), and incubate at 4°C for 10 min.
d. Shock the cells by centrifugation at 4°C17200 g (12000r/min Sorvan SS-34 turntable) for 10 min, and add lOmmol/L Tris-Cl (pH7.1) to the supernatant until pH 7.1 is reached.
e. The supernatant is filtered through a 100-ml secondary Nalgene filter (0.45-um cellulose nitrate membrane), and the filtrate is dialyzed through a 100-fold volume of 10 mmol/L Tris-Cl (pH 7.1) at 4°C. Continuing with step 4, purify the fusion protein from the supernatant by straight chain starch agarose chromatography.
Purification of fusion protein
4. The supernatant obtained in step 3 is loaded onto the column, rinsed with 100 ml of 10 mmol/L Tris-Cl (pH 7.1), and washed with 100 ml of column wash buffer.
5. Elute the bound fusion proteins with 50 ml of column elution buffer and collect them in steps of 1 ml each.
6. Analyze the distribution of the fusion proteins by SDS polyacrylamide gel electrophoresis, combine the samples containing the fusion proteins and store at -70°C.
7. The fusion proteins are cleaved with an appropriate protease to release the target proteins.
a. Add thrombin, enterokinase, or Factor Xa (chosen according to the site in the fusion protein). Add 50 units of protease dissolved in lml PBS per ml of resin. Mix by inverting the tube several times and shaking at room temperature for 2-16 h. Determine the time required to obtain the highest yield using a small-scale experiment.
b. Centrifuge at 500 g (2100 r/min Sorvall SS-34 head) for 5 min at 4°C and carefully transfer the supernatant to a new tube.
Separate the target protein from the protease by conventional chromatography or SDS polyacrylamide gel electrophoresis. 