pMal-pIII Vector Protocol
- Prepare double-stranded (RF) phage DNA from each selected Ph.D. clone using standard procedures, e.g. Sambrook, (3rd ed.), (pp 3.23-3.25). Alternatively, a cassette containing the insert sequence can be obtained by PCR of single stranded phage DNA with M13 extension primer (NEB #E8101) and -96gIII sequencing primer (NEB #S1259).
- Digest DNA with Acc65I and EagI in NEBuffer 3. Isolate small fragment (52-67 bp), containing selected peptide sequence and flanking leader sequence, by nondenaturing polyacrylamide or high-resolution agarose gel electrophoresis.
- Ligate purified segment into Acc65I/EagI digested pMal-pIII vector.
- Transform into suitable host, e.g. E. coli TB1 (NEB #E4122) or NEB Turbo Competent E.coli (NEB #C2984H).
- Carry out pilot scale expression experiments. Refer to pMal Protein Fusion and Purification System manual page 17. To purify the fusion on amylose resin (NEB #E8021 ) based on the pocedure on p.23 for a 1L culture:
a. Harvest cells by centrifugation at 4000 x g for 20 min. and discard the supernatant. Resuspend cells in 400 ml 30 mM Tris-HCl, 20% sucrose, pH 8.0 (80 ml for each gram of cells wet weight). Add EDTA to 1 mM and incubate for 5-10 min. at room temperature with shaking or stirring.
b. Centrifuge at 8000 x g for 20 min. at 4°C. Remove all the supernatant. Resuspend the pellet in 400 ml of ice-cold 5 mM MgSO4. Shake or stir for 10 min. in an ice bath.
c. Centrifuge at 8000 x g for 20 min. at 4°C. The supernatant is the cold osmotic shock fluid.
d. Add 8 ml 1 M Tris-HCl, pH 7.4 to osmotic shock fluid.
e. Continue to amylose resin affinity purification (step 7, p. 24).