Protein Engineering Protocols - Mycobacteriology research center

Synthesis of Libraries and Screening With the DHFR PCA 2558. Buffer A: 6 M guanidinium-HCl (Gdn-HCl), 0.1 M NaH 2PO 4, and 0.01 M Tris-HCl,pH 8.0; supplemented with 10 µM phenylmethyl sulfonyl fluoride, 7.2 mMβ-mercaptoethanol, 15–25 mM imidazole, and 300 mM NaCl.9. Buffer B: same as buffer A, but pH 6.3 and supplemented with 7.2 mMβ-mercaptoethanol and, sometimes, 15 mM imidazole.10. Buffer E: 4 M Gdn-HCl and 0.025 M NaOAc, pH 4.5.11. Dithiothreitol (DTT).12. 6 M KOH.2.7. Preparation of SS320 and BL21 pREP4 pQE-32 ras-F [3]Electrocompetent Cells1. Overnight (O/N) preculture of SS320 or BL21 pREP4 pQE-32 ras-F [3].2. 500 mL SOB medium: 2% tryptone, 0.5% yeast extract, 1 mL 10 mM NaCl, 2.5 mMKCl, 10 mM MgCl 2, and 10 mM MgSO 4; supplemented with 0.2% glucose forSS320 strain.3. 500 mL LB medium supplemented with 0.2% glucose for BL21 pREP4 strain.4. 2 L of ice-cold autoclaved deionized water.5. 10% autoclaved glycerol solution (Bioshop).2.8. Preparation of XL-1 Blue and BL21 pREP4 Chemiocompetent Cells1. O/N preculture of XL-1 Blue or BL21 pREP4.2. 500 mL SOB medium supplemented with 0.2% glucose for SS320 strain.3. 500 mL LB medium supplemented with 0.2% glucose for BL21 pREP4 strain.4. Transformation buffer: 10 mM Pipes, 15 mM CaCl 2and 250 mM KCl, pH 6.7,with KOH. After the pH is set, MnCl 2is added to a concentration of 55 mM.5. Dimethylsulfoxide.3. Methods3.1. General Considerations3.1.1. Steric Requirements in PCAThe spatial orientation of the PCA fragments is crucial to whether the PCAreporter protein can fold from its cognate fragments, and is determined by theorientations of the amino or carboxyl termini of the interacting proteins in thecomplex formed (see Fig. 1 for schematization of spatial considerations encounteredin designing linkers). In the design of the protein–PCA fragment fusions,it is, therefore, important to determine, a priori, whether the fragments would bebrought together by a given combination of fusion constructs in such a way thatthe topology of the native structure could be achieved from a given configurationof the fusions. The two main factors that will determine whether correctfolding can be attained are the orientation of the fusion (carboxyl- and/or aminoterminal)and second, the length of polypeptide linkers between the individualfragments and the proteins to which they are fused. Our experience has shown that