Protein Engineering Protocols - Mycobacteriology research center

Monobodies 9910. Agarose-phosphate solution: mix 8 mL water, 2 mL 0.5 M potassium phosphatebuffer, pH 7.0, and 0.07 g agarose per plate (multiply these amounts according tothe number of plates).11. 2X β-galactosidase assay solution: 120 mM Na 2HPO 4, 80 mM NaH 2PO 4, 20 mMKCl, 2 mM MgSO 4, 0.54% β-mercaptoethanol, 0.0008% sodium dodecyl sulfate,8 mg/mL 2-nitrophenyl-β-D-galactoside, pH 7.0. Store in small aliquots at –20°C.12. Buffer B: 20 mM Tris-HCl, pH 8.0, and 500 mM NaCl.13. Buffer C: 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 500 mM imidazole.14. Buffer D: 50 mM sodium phosphate and 500 mM NaCl, pH 8.0.15. Tris-HCl–EDTA (TE) buffer: 10 mM Tris-HCl and 1 mM EDTA, pH 8.0.16. 50X Tris–acetate–EDTA buffer: dissolve 242 g Tris-base, 57.1 mL glacial aceticacid, and 18.6 g EDTA in a final volume of 1 L ddH 2O. Adjust pH to 8.3.17. Escherichia coli strains: CJ236 (Bio-Rad), KC8 (Origene), XL-1 Blue (Stratagene),and SS-320 (ref. 11; see Note 1).18. Yeast strains: EGY48 and RFY206 (Origene; refs. 12 and 13).2.3. Vectors1. pAS38: a derivative of the pBluescript SK(+) phagemid (Stratagene) encodingFnfn10 fused to the C-terminal domain of M13 gene III. We constructed theFNfn10 gene using preferred codons for E. coli (8). This is for monovalent displayof monobodies. The PvuII fragment in this vector is in the opposite direction fromthat in pBluescript SK(+).2. pAS45: a monobody expression plasmid derived from pET15b (Novagen). Itexpresses a His-tag–monobody fusion protein. The monobody gene contains anArg6 to Thr mutation that was originally introduced to eliminate a thrombin cleavagesite (8).3. pAS47: A derivative of pAS38 containing a stop codon in the FG-loop (Fig. 1A).This is used as the template for mutagenesis for the construction of libraries inwhich residues in the FG-loop are diversified.4. JCFN: A derivative of JC-M13-88 (14) encoding a monobody–gene VIII fusionprotein. This is an M13 phage system for multivalent display of monobodies.5. pYT45: A derivative of pYesTrp2 (Invitrogen) encoding B42 (activation protein)–monobodyfusion protein (10,15). This is used to construct monobodylibraries for yeast two-hybrid screening (see Note 2).6. pSH18-34: a LacZ reporter plasmid for yeast two-hybrid screening (Origine).7. pEG202: LexA-bait plasmid for yeast two-hybrid screening (Origene; see Note 2).The monobody vectors and libraries are available from the correspondingauthor.3. Methods3.1. Library ConstructionWe used the Kunkel mutagenesis technique to construct most of our libraries(16). We used the “top” end (BC, DE, and FG loops; Fig. 1A) of FNfn10 as the