PurificationofMBP(maLTosebindingproteins)FusedProteins

Express fusion proteins as per the GST-fused protocol up to Step 7 (Day 3). All steps in protein purification should be done at 4° C unless otherwise stated.

1. Pre-wash 10-20 ml of New England Biolabs amylose resin with 20 mM Tris pH7.4, 200 mM NaCl, 10 mM b ME, 1mM EDTA.

2. Pellet the cellular debris (SS34, 13000 rpm, 15’) from the lysate (Step 7).

3. Dilute the supernatant to 100 ml with 20mM Tris pH7.4, 200 mM NaCl, 10 mM b ME, 1mM EDTA and apply to amylose resin. Mix 1 hour.

4. Resuspend pellet in 100 ml buffer for analysis on SDS-PAGE.

5. Elute "unbound" proteins from amylose resin by washing resin with 10 column volumes of 20 mM Tris pH7.4, 200 mM NaCl, 10mM b ME, 1 mM EDTA.

6. Elute bound MBP fusion protein with 10 fractions of 10 mM maltose in 20 mM Tris pH 7.4, 200 mM NaCl, 10 mM b ME, 1 mM EDTA or in some other buffer of choice, depending on subsequent purification steps. If the fusion protein is to be cleaved with factor Xa as the next step, it is preferable to elute in a buffer at pH 8, without EDTA.

7. Check eluted fractions for protein by SDS-PAGE or protein assay. Combine appropriate fractions.

8. Factor Xa cleavage. Add CaCl2 to 2 mM final conc., then add 5-10 units of factor Xa per ml of eluted fusion protein (assuming eluted protein is approximately 1 mg/ml). Monitor digestion over hours/days by SDS-PAGE. This step could be done at room temperature to speed up proteolysis, depending on the stability of the protein of interest.