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Constructing a Random-Peptide Library for Phage Display
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
The protocol describes the construction of a large (108 to 1010) primary random peptide phage library in fUSE5 or f88-4 strains. The library requires approximately 100 μg of cut vector with a roughly equimolar amount of degenerate insert in a total volume of 20 ml, followed by electroporation into electrocompetent cells. Amplification of an already existing library is described in detail in the protocol on library amplification.
Procedure
A. Preparation of the Vector and Insert
Preparation of fUSE5-SfiI, including cleavage and removal of the stuffer between the two SfiI restriction sites by ultrafiltration through a Centricon 100, is described in Protocol ID#2171.
Preparation of f88-4-PstI , including cleavage and removal of the stuffer between the two PstI restricition sites by Isopropanol precipitation, is described in Protocol ID#2163.
Preparation of a degenerate oligonucleotide insert encoding a random peptide library is described in Protocol ID#2172.
B. Ligation
The concentration of cut vector should not exceed 5 μg/ml during the ligation in order to prevent the formation of multimers (containing two or more vector molecules). A large library requires approximately 100 μg cut vector, corresponding to 20 ml of ligation reaction solution. A 20 ml scale is assumed in the following protocol (make adjustments as necessary if different amounts are used).
1. Mix the following in a suitable vessel (concentrations listed are the final reaction concentration):
5 μg/ml (823 μM) of Vector DNA
1 to 4 moles insert per mole Vector DNA of Insert DNA
10 Weiss Units/ml of Ligase
1X of Ligase Buffer (30 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 500 μM ATP)
25 μg/ml Acetylated BSA
2. Incubate at 16°C for approximately 5 hr (see Hint #1).
3. Combine 20 μl of the Ligation Mixture with 3.3 μl of 70/75/BPB solution and mix well. Freeze the remainder of the Ligation Mixture while waiting for the gel electrophoresis results.
4. Electrophorese the aliquot from the Ligation Mixture on a 0.8% Agarose/4X GBB minigel (see Protocol ID#455; also see Hint #2).
5. Continue with the procedure if the results from the Electrophoresis of the Ligation Mixture are positive (see Hint #3).
6. For every ml of frozen Ligation Mixture, add 100 μl of 3 M Sodium Acetate (2 ml for a 20 ml ligation) and 50 μl of 250 mM EDTA (1 ml for a 20 ml ligation, see Hint #4).
7. Allow the mixture to thaw, gradually mixing the Sodium Acetate / EDTA solution into the thawing slurry.
8. Divide the Ligation Mixture solution evenly into 30 ml Corex glass centrifuge tubes (see Hint #5).
9. Add 2 volumes of 100% Ethanol to each tube, cover with Parafilm, and invert the tube to mix (see Hint #6).
10. Incubate the mixture overnight at 4°C.
11. Centrifuge at 10,000 rpm for 20 min in a Sorvall™ SS-34 rotor (12,000 X g) at 4°C (see Hint #7).
12.Discard the supernatant without disturbing the DNA pellet.
13. Centrifuge again at 10,000 rpm for 10 min in a Sorvall™ SS-34 rotor (12,000 X g) at 4°C (see Hint #8).
14. Using a pipette, carefully remove and discard any remaining supernatant without disturbing the DNA pellet.
15. Dry the DNA pellet briefly under vacuum.
16. Dissolve and pool all of the DNA precipitates in a total of 2 ml of TE Buffer.
17. Load the DNA sample onto a Centricon-100 KDa ultrafilter (Amicon) and concentrate the sample by centrifuging at 2,500 rpm for approximately 40 min in a Sorvall™ SS-34 rotor (750 X g) at room temperature (see Hint #9).
18. Discard the pass-through volume from the Centricon-100.
19. Add 2 ml of TE Buffer to the Centricon-100 well and centrifuge until the stop-volume is reached.
20. Repeat Step #B18 to Step#B19 two more times (for a total of three washes).
21. Discard the pass-through volume from the Centricon-100.
22. Collect the retentate by back-centrifugation as described in Amicon's instructions.
23. Transfer the retentate into a 500 μl microcentrifuge tube and estimate the total collected volume with a pipette (should be approximately 100 μl).
24. Add a volume of TE Buffer to the Centricon-100 that is equivalent to the difference in volume between the collected retentate volume and 180 μl (see Hint #10).
25. Vortex the Centricon-100 to thoroughly wash the membrane and then collect the wash by back-centrifugation as described in Amicon's instructions.
26. Transfer the retentate into the same 500 μl microcentrifuge tube and estimate the total collected volume with a pipette (should be 180 μl).
27. Use twelve 15 μl portions of this solution to electroporate twelve 200 μl portions of electrocompetent cells as described in Protocol ID#2186 (see Hint #11).
28. Virions in the supernatant of the resulting four 1-liter cultures represent the library.
29. Purify virions from these cultures according to Protocol ID#2177 (use modifications that omit detergent treatment, also see Hint #12).
30. Sequence a sample of the purified phage to confirm the degenerate region of the coding sequence.
Solutions
IPTG Solution
0.2 M IPTG
Filter sterilize and store at 4°C 
TE Buffer
Autoclave and store at room temperature
10 mM Tris-HCl, pH 8.0
1 mM EDTA
3 M Sodium Acetate
GBB (40X)
Store at room temperature
Adjust the final volume to 700 ml
Dissolve in approximately 500 ml water
45.94 g Anhydrous Sodium Acetate (or 76.16 g Trihydrate)
142.4 g Tris
Adjust pH to 8.3 with Glacial Acetic Acid
18.83 g Disodium EDTA-2H2O
70/75/BPB Solution
70% (v/v) Glycerol
75 mM EDTA
0.3% (w/v) Bromophenol Blue
Acetylated BSA
25 μg/ml Acetylated Bovine Serum Albumin
Ligase Buffer (10X)
300 mM Tris-HCl, pH 7.8
5 mM ATP
100 mM MgCl2
100 mM DTT
Ligase
10 Weiss Units/ml Ligase
Insert DNA
1 to 4 moles Insert DNA per mole Vector DNA
Vector DNA
Not to exceed 5 μg/ml
BioReagents and Chemicals
IPTG
Oligonucleotide
DNA Ligase
ATP
Glycerol
EDTA
Glacial Acetic Acid
Magnesium Chloride
Sodium Acetate
DTT
Tris-HCl
Bovine Serum Albumin, Acetylated
Bromophenol Blue
Protocol Hints
1. For restriction sites with 4 basepair overhangs, such as those generated in f88-4 by HindIII and PstI. Incubation at 10°C may be better for 3 basepair overhangs, such as those generated in fUSE5 by SfiI.
2. Be sure to electrophorese appropriate DNA markers, such as λ-BstEII or λ-HindIII. Use 4X GBB to prepare the gel and add 1 μg/ml Ethidium Bromide when the gel solution has cooled to below 50°C. Use 1X GBB containing 1 μg/ml Ethidium Bromide for electrophoresis.
3. The intended ligation product is unit-length covalently closed circular (cccDNA) DNA molecules containing 1 molecule of vector spliced at both ends to one molecule of insert. These cccDNAs are a mixture of approximately 7 different topoisomers that have very little supercoiling, and which separate under ordinary electrophoresis conditions. The addition of Ethidium Bromide to the gel introduces positive supercoils into the cccDNA in sufficient density to cause all topoisomers to co-migrate as an approximately 5 Kbp linear double-stranded fragments (this is also where the negatively supercoiled Replicative Factor molecules, which exist naturally in the infected cell, migrate). The contributor of this protocol finds that a ligation yielding approximately 10% of such a form) produces excellent libraries. Often, good libraries are obtained even if no cccDNAs are detectable, provided some open-circular products (co-migrates with approximately 11-Kbp linear double-stranded fragments) are apparent. If the quality of the library is unclear, it is recommended to do a small-scale electroporation with a diluted sample representing 0.1 μl (0.5 ng) of the ligation mixture to transfect 50 μl electrocompetent cells as described in steps 18-27 of Protocol ID#2186; a good ligation will yield approximately 107 transfectants/μg (5,000 transfectants per 0.5 ng).
4. The final EDTA concentration should be a 1.25-fold excess over the current Mg++ concentration. The total volume should now be 23 ml.
5. Do not exceed 7 ml per Corex tube. Use 5.75 ml per Corex tube for a 20 ml ligation reaction.
6. Add 11.5 ml of 100% Ethanol per 5.75 ml of aqueous solution.
7. After placing the tubes into the rotor, mark the top edge of the tube indicating the approximate position of the pellet (pellets form on the side of the tube furthest from the center of the rotor). In glass tubes, the precipitated DNA forms a thin coat along the tube that can be very difficult to identify. Marking the approximate location of the tube where the pellet will form aids in identifying the DNA pellet.
8. Align the tubes into the rotor exactly as they were in the first centrifugation step by using the marks on the top edge of the tube.
9. The time to reach the stop-volume will depend on the concentration of the sample. The manufacturer's instructions on the Centricon-100 will have all the details.
10. For example, if the collected retentate from Step #B23 was 110 μl, then the volume to add to the Centricon-100 in Step #B24 would be 70 μl.
11. For libraries in f88-4 vector, be sure to add IPTG Solution to 1 mM if there is a need to induce the recombinant gene VIII. The IPTG is not necessary in MC1061 host cells that have no LacI gene.
12. It is not necessary to purify library virions to the high degree achieved in the recommended protocol; doubly-PEG-precipitated virions are adequately pure. However, for a valuable general-purpose library for use over many years, proceed with the remaining steps of the recommended protocol to ensure highly purified virions. These should be stable for decades.
