Protein Engineering Protocols - Mycobacteriology research center

208 Sidhu et al.2.2. Selection and Analysis of P8 Variants That Increase FusionProtein Display2.2.1. Selection of Phage From the hGH-P8 Library1. 0.2% bovine serum albumin (BSA) in PBS.2. 100 mM HCl.3. 1.0 M Tris-base.4. 96-well maxisorp immunoplates (NUNC, Roskilde, Denmark).5. E. coli XL-1 Blue (Stratagene, La Jolla, CA).6. PBS-T buffer: PBS and 0.05% Tween-20.7. PBS-T-BSA buffer: PBS, 0.05% Tween-20, and 0.2% BSA.8. 2YT/carb/kan medium: 2YT, 50 µg/mL carbenicillin, and 25 µg/mL kanamycin.2.2.2. Phage Enzyme-Linked Immunosorbent Assay Screento Assess Levels of hGH Display1. 1.0 M H 3PO 4.2. 2YT/carb/kan medium (see Subheading 2.2.1.).3. 3,3′,5,5′-tetramethylbenzidine/H 2O 2peroxidase (TMB) substrate (Kirkegaard & PerryLaboratories Inc., Gaithersburg, MD).4. 96-well maxisorp immunoplates (see Subheading 2.2.1.).5. Carbenicillin (see Subheading 2.1.1.).6. E. coli XL-1 Blue (see Subheading 2.2.1.).7. Horseradish peroxidase/anti-M13 antibody conjugate (Amersham-Pharmacia).8. Kanamycin (see Subheading 2.1.1.).9. LB/tet plate: LB agar and 5 µg/mL tetracycline.10. M13KO7 helper phage (see Subheading 2.1.1.).11. PBS (see Subheading 2.1.1.).12. PBS-T buffer (see Subheading 2.2.1.).13. PBS-T-BSA buffer (see Subheading 2.2.1.).14. PEG/NaCl (see Subheading 2.1.1.).15. Tetracycline (see Subheading 2.1.4.).3. MethodsThe methods outlined below describe the design (Subheading 3.1.), construction(Subheading 3.2.), selection (Subheading 3.3.1.), and screening(Subheading 3.3.2.) of P8 libraries to identify mutations that improve proteindisplay levels. We describe protocols for the improved display of hGH usingthe previously described phagemid, pS1607, but the methods are applicable toany protein of interest. The only modifications are the use of a phagemiddesigned to display the protein of interest in place of pS1607, and the use of aligand that binds the protein of interest with high affinity in place of hGH bindingprotein (hGHbp).