Protein Engineering Protocols - Mycobacteriology research center

Monobodies 1073.4.1. Cloning of a Monobody1. Amplify the gene of a monobody of interest using oligonucleotides NdeMetThrFNF(5′-CGGGATCCCATATGCAGGTTTCTGATGTTCCGACCGACCTG-GAAGTTGTTGCTG-3′; this contains the Arg6 to Thr mutation) and FNGKKGKR(5′-CCGACTCGAGTTACTATTTACCTTTTTTACCGGTACGGTAGTTAATC-GAG-3′), then digest with NdeI and XhoI and clone in pET15b (Novagen) digestedwith the same enzymes. Confirm the gene in the expression vector by sequencing.3.4.2. Expression of a MonobodyThis protocol is for a 100 mL culture, which should yield sufficient quantitiesof a monobody for initial characterization.1. Transform BL21(DE3) cells (Novagen) with a monobody expression vector(see Note 9).2. Inoculate 10 mL M9-tryptone-Ap with transformed cells and incubate the culturewith vigorous shaking at 30°C for 10 to 16 h.3. Inoculate 2 mL of the preculture to prewarmed 100 mL M9-tryptone-Ap, andincubate with vigorous shaking at 37°C. When the OD 600nmreaches 0.7, add IPTGto a final concentration of 0.5 mM.4. Incubate with vigorous shaking at 37°C for 3 h more, and harvest the cells by centrifugation.The cells can be stored at –20°C until use.3.4.3. Purification of a MonobodyThis protocol is for cells from 100 mL culture (Subheading 3.4.2.)1. Suspend the cell pellet in 6.4 mL of 50 mM Tris-HCl, pH 8.0.2. Add 64 µL of 50 mg/mL lysozyme and 128 µL of 50 mM PMSF. Mix and incubateat 37°C for 15 min. Sonicate the suspension until it is no longer highly viscous.3. Add 910 µL of 4 M NaCl. Centrifuge at 27,000g (15 krpm, SS34 rotor) for 10 minat 4°C. Collect the supernatant.4. Apply protein solution in a Hi-Trap chelating column (1 mL; Amersham-Pharmacia Biotech) that has been loaded with 0.1 M NiCl 2and equilibrated withbuffer B. Wash the column with 6 mL of buffer B, then with 6 mL of buffer B containing60 mM imidazole.5. Elute the monobody with 6 mL of buffer C. Collect 0.5 to 1 mL aliquots of theeluent in microcentrifuge tubes. Analyze the eluent with sodium dodecyl sulfatepolyacrylamide gel electrophoresis.3.4.4. Biotinylation of a MonobodyWe conjugate biotin at the Lys amino group. Although this is not site specific,the Lys cluster in the C-terminal extension should be a preferred modificationsite. This method can be used to conjugate other moieties, e.g., afluorescent dye.