Synthesis of degenerated libraries of the ras-binding domain of raf and rapid selection of fast-folding and stable clones with the dihydrofolate reductase protein fragment complementation assay (original) (raw)

Abstract

The protein-engineering field is mainly concerned with the design of novel enzyme activities or folds and with understanding the fundamental sequence determinants of protein folding and stability. Much effort has been put into the design of methods to generate and screen libraries of polypeptides. Screening for the ability of proteins to bind with high affinity and/or specificity is most often approached with phage display technologies. In this chapter, we present an alternative to phage display, performed totally in vivo, based on the dihydrofolate reductase (DHFR) protein-fragment complementation assay (PCA). We describe the application of the DHFR PCA to the selection of degenerated sequences of the ras-binding domain (RBD) of raf for correct folding and binding to ras. Our screening system allows for enrichment of the libraries for the best-behaving sequences through iterative competition experiments, without the discrete library screening and expansion steps that are necessary ...

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References (90)

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  2. Restriction enzymes: HpaI, XmaI, EcoNI, and XbaI (NEB or Fermentas).
  3. XL-1 Blue chemiocompetent cells (see Subheading 2.8.).
  4. Clones Competition Experiment 1. Glass culture or 15-mL conical tubes (Corning).
  5. Solid and liquid selective medium (same protocol as in Subheading 2.3., item 6, except that agar is not added for the liquid medium).
  6. Plasmid Midi Kit (Qiagen) or alkaline lysis maxiprep.
  7. LB medium supplemented with 100 μg/mL ampicillin and 25 μg/mL kanamycin.
  8. 5. Isolation of Clones and Sequencing 1. Restriction enzymes: HpaI, XmaI, EcoNI, and XbaI (NEB or Fermentas).
  9. XL-1 Blue competent cells.
  10. 24-well plates (Corning), LB-agar with 100 μg/mL ampicillin.
  11. 2-mL V-shaped 96-well culture block (VWR).
  12. Montage™ Plasmid Miniprep 96 kit (Millipore, LSKP 096 01) or smaller scale prep kit, such as QIAprep™ Spin Miniprep Kit (27104) (Qiagen), depending on the number of samples to be processed.
  13. Vacuum manifold Multiscreen Resist™ (Millipore, MAVM 096 OR).
  14. Oligonucleotide primer for sequencing specific to the plasmid harboring the library (IDT).
  15. Appropriate restriction endonucleases (SalI and XhoI, in this case) and reagents necessary for ligation (see Subheading 2.2.).
  16. BL21 pREP4 competent cells and LB with 100 μg/mL ampicillin and 25 μg/mL kanamycin petri dishes.
  17. Terrific broth (TB) medium (12 g tryptone, 24 g yeast extract, 4 mL glycerol, 2.31 g KH 2 PO 4 , and 12.54 g K 2 HPO 4 ; add water to a final volume of 1 L) supplemented with 100 μg/mL ampicillin and 25 μg/mL kanamycin.
  18. 50-mL conical tubes (Corning).
  19. A centrifuge and rotor that accommodate 96-well plate, such as Eppendorf 5810 or 5810 R and A-4-62, respectively.
  20. Ni-NTA Spin Kit or Ni-NTA Superflow™ 96 Biorobot Kit (Qiagen), depending on the number of samples to be processed. An affordable alternative to the Superflow 96 Biorobot Kit is the following: we use Ni-NTA Superflow resin (Qiagen), 0.25-mm glass fiber filter 96-well plates (3510), 0.2-μm polyvinylidene fluoride (PVDF) membrane 96-well plates (3504), 96-well volume extender (3584), and fraction collector (3958) from Corning.
  21. Vacuum manifold Multiscreen Resist™ and a large collection and sealing block (Millipore; respectively, MAVM 096 OR and OT).
  22. Chill cells in an ice-water bath 10 to 15 min and transfer to a prechilled 1-L cen- trifuge bottle.
  23. Centrifuge cells for 20 min at 5000g.
  24. Pour off supernatant and resuspend the pellet in 5 mL of ice-cold water. Add 500 mL of ice-cold water and mix well. Centrifuge cells as in step 4.
  25. Pour off supernatant immediately and resuspend the pellet by swirling in the remaining liquid.
  26. Add another 500 mL of ice-cold water, mix well, and centrifuge again as in step 4.
  27. Pour off supernatant immediately and resuspend the pellet by swirling in the remaining liquid.
  28. If fresh cells are to be used for electroporation, place suspension in a prechilled, 50-mL polypropylene tube, and centrifuge for 10 min at 5000g and 2°C. Estimate the pellet volume (usually ~500 μL from a 500-mL culture) and add an equal vol- ume of ice-cold water to resuspend cells (on ice). Aliquot 50 to 300 μL cells into prechilled microcentrifuge tubes. The cell density is approx 2 × 10 11 cells/mL.
  29. If frozen cells are to be used for electroporation, add 40 mL of ice-cold 10% glyc- erol to the cells from step 8 and mix well. Centrifuge cells as described in step 9. Estimate the pellet volume and add an equal volume of ice-cold 10% glycerol to resuspend cells (on ice). Place 50 to 300 μL cells into prechilled microcentrifuge tubes and freeze on dry ice. Store at -80°C.
  30. 9. Preparation of XL-1 Blue and BL21 pREP4 Chemiocompetent Cells Cells were prepared according to H. Inoue et al. (49), with one slight modi- fication: the cells are washed only once after the first centrifugation step. The bottles containing the cell pellets are placed, inverted, on a piece of paper at 4°C to remove most traces of medium (see Note 29).
  31. Inoculate 200 mL of SOB or LB medium in a 2-L flask from an O/N culture of the respective cell strain, and grow to an OD 600 of 0.6 at 18°C with vigorous shak- ing (200-250 rpm).
  32. Chill cells on ice for 10 min and transfer the culture to a 500-mL centrifuge bottle.
  33. Centrifuge cells at 2500g for 10 min at 4°C.
  34. Resuspend the pellet in 80 mL of ice-cold transformation buffer, incubate in an ice bath for 10 min, and centrifuge as in step 3.
  35. Gently resuspend the cell pellet in 20 mL of transformation buffer, and add DMSO with gentle swirling to a final concentration of 7%.
  36. Incubate in an ice bath for 10 min.
  37. Aliquot the cell suspension, and freeze in liquid nitrogen.
  38. Notes
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