NPLconstruct.doc Page 1 11/14/18

METHODS SUMMARY

Solutions, media and protocols for constructing and working with phage-display libraries are described on our laboratory’s world wide web site, In addition, anyone wishing to request materials or information from our laboratory can contact us by fax at 573-882-0123.

NPL Construction

A NPL is constructed by ligating randomly fragmented genomic DNA with linkers containing restriction enzyme recognition sites. The linker-ligated genomic DNA fragments and vector are then digested with appropriate restriction enzymes, ligated, electroporated into E. coli cells, and propagated.

The following protocol contains references (indicated as underlined text) to the names of documents accessible from our world wide web site, which contain specific reagent recipes and protocols.

Random fragmentation of insert DNA by DNAseI

  1. Perform a pilot experiment to determine the appropriate concentration of DNAseI necessary to digest genomic DNA to a fragment size range of approximately 100–1000 base pairs.
  2. Prepare fresh 10× DNAseI buffer

500 mM Tris pH 7.8

10 mM MnCl2

1 mg/ml nuclease-free BSA

2.2.Prepare a series of six digestion tubes containing 25 µl of 100 µg/ml genomic DNA in 1× DNAseI buffer, on ice.

2.3.Prepare six 3:4 fold serial dilutions of DNAseI (Boehringer-Mannheim; molecular biology grade) from 1.0 to 0.237 U/ml in 1× DNAseI buffer. NOTE: these concentrations may need to be adjusted depending on the source and purity of the DNA to be digested.

2.4.Add 8.75 µl of diluted, ice cold DNAseI to each of the digestion tubes, vortex gently, immediately transfer to a 15º water bath, and incubate for exactly 10 min.

2.5.Stop the reactions by adding 4 µl 250 mM EDTA (stdpreps.doc) and heating 10 min at 68º.

2.6.Add 5.6 µl 70/75BPB (stdpreps.doc) to each digestion and run 14 µl on a 7% (19:1 monomer:bisacrylamide) polyacrylamide gel (1× TBE; 1 mm thickness) to determine fragment size range, using standard procedures (22).

  1. Based on results of the pilot experiment, perform a scaled-up DNAseI digestion of 50–100 µg DNA (at a concentration of 100 µg/ml, as in the pilot experiment) following exactly the procedures described in Section 1, and using the appropriate DNAseI concentration to yield fragments of ~100–1000 base-pairs. Run 12 µl of the digested DNA (plus 2 µl 70/75/BPB) on a 7 % polyacrylamide gel as described in Section 1.6, to confirm that the DNA fragments fall approximately within the correct size range.
  2. Dilute the digested DNA ~6-fold in water, extract with neutralized phenol (stdpreps.doc) and chloroform, dialyze in a Slide-a-Lyzer (Pierce; 10,000 mw cut-off; 3–15 ml sample volume) against four changes of 500 ml TE (stdpreps.doc), concentrate to ~100 µl on a Centricon 30-kDa ultrafilter (Amicon), and quantify by spectrophotometry. A typical yield is at least 70% the quantity of starting material.

Blunt-end polishing of fragmented DNA

  1. Polish 50–100 µg DNAseI digested DNA fragments, at a concentration of ~120 µg/ml, with T4 DNA polymerase (Boehringer Mannheim, ~2 U per g DNA) in the presence of 500 M dNTPs and buffer (final concentration = 50 mM Tris-HCl, 15 mM (NH4)2SO4, 7 mM MgCl2, 0.1 mM EDTA , 10 mM ß-mercaptoethanol, 0.2 mg/ml BSA, pH 8.8), at 15º for 1 hour. After 10 min incubation at 75º to inactivate T4 DNA polymerase, cool the reaction mixture to room temp, add ~1.67 U DNA polymerase I Klenow fragment, exonuclease minus (Promega) per µg DNA, and incubate at 37º for 1 hour. Extract with neutralized phenol and chloroform, vacuum evaporate for ~30 min to remove residual chloroform, then wash with 5–10 ml TE and concentrate to ~80 l on a Centricon 30-kDa ultrafilter (Amicon), and quantify by spectrophotometry. The final yield is typically ~2/3 the quantity of starting material.

Preparation of linker-ligated insert DNA

Figure 3. Hairpin linkers used in NPL library construction.

A) Hind III linker. B) PstI linker.

  1. Polished insert DNA fragments are ligated to blunt-end, hairpin linkers (HindIII and PstI, panels A and B, Figure 3) containing restriction enzyme sites for insertion into the f88 vector, and appropriate coding sequence to preserve the gVIII reading frame and leader sequence.
  2. Ligate HindIII and PstI linkers (5-fold molar excess termini) to ~30 µg polished DNA fragments, at a concentration of ~120 µg/ml, overnight at 16º in buffer (final concentration = 66 mM TrisHCl, 5 mM MgCl2, 1 mM DTE, 1 mM ATP, pH 7.5) with 0.75-1.0 Weiss Units T4 DNA ligase (Boehringer Mannheim) per g DNA fragments.
  3. After 15 min incubation at 68º to inactivate the ligase, add 1/10 volume 10× buffer (500 mM TrisHCl, 100 mM MgSO4, 1 mM DTT) and 0.75–1.0 U exonuclease III per g original DNA fragments directly to the ligation mixture. Incubate at 37ºfor 30 min. This step digests unligated DNA fragments without harming linker-ligated DNA fragments, which are protected by the hairpin linkers.
  4. Add EDTA to a final concentration of 8.3 mM, 1/10 volume 10X buffer (670 mM KH2PO4, 100 mM ß-mercaptoethanol, pH 7.9) and 1 U exonucleaseVII (GibcoBRL) per g original DNA fragments. Incubate at 37º for 90 min. This step removes residual single stranded DNA generated by the exonuclease III digestion.
  5. Extract the remaining double stranded, linker-ligated DNA once with neutralized phenol and twice with chloroform; vacuum evaporate briefly, concentrate on a Centricon 30-kDa ultrafilter (Amicon), and quantify by spectrophotometry. A typical yield is ~1.5× the quantity of original DNA fragments.

Final preparation of insert DNA

  1. Digest insert DNA (~40 µg), with 100 U of restriction enzymes HindIII and PstI per µg DNA fragments in buffer (final concentration = 6 mM TrisHCl, 6 mM Mg Cl2, 50 mM NaCl, 1 mM DTT, pH 7.5) supplemented with 1 mg/ml BSA, in a total volume of 5 ml at 37º for 4–18 hours. Extract the DNA once with neutralized phenol, twice with chloroform, then wash with 5–10 ml TE and concentrate to ~80 l on a Centricon 30-kDa ultrafilter (Amicon).
  2. To remove short fragments released by restriction digestion, purify the restriction digested DNA fragments by polyacrylamide gel electrophoresis on a 7% gel (19:1 monomer:bisacrylamide; 1× TBE; 1 mm thickness) in a single preparative lane (8.5 cm wide) using standard procedures (22). Excise the portion of the gel containing insert DNA 65 bp or longer, and recover the DNA by electroelution. For this purpose we use 4-6 Centricon 30-kDa ultrafilters and an Amicon Centrilutor apparatus, following the manufacturer’s instructions.

Preparation of Primary Library

  1. We chose the f88 vector (Choukri, Sam, personal communication; libeseq.doc) for NPL construction to facilitate display of large inserts; the vector contains a wild-type gVIII as well as a recombinant gVIII, along with restriction enzyme cloning sites (for more information see vectors.doc). Since both wild type (~3900 copies) and recombinant (~150 copies) gVIII proteins (pVIII) are incorporated into the phage coat during assembly, phage survival is ensured by the presence of wild type coat protein, allowing peptide fusions that would otherwise impair coat-protein function to be displayed, and thereby accommodating the larger inserts desired for the NPL.
  2. Prepare f88 vector RF (RFmaxiprep.doc) and cleave with HindIII and PstI (f88cleavage.doc). Treat the cleaved vector with calf intestinal alkaline phosphatase (CIP) by standard procedures (22). This step is not absolutely necessary, but we have found that this treatment substantially reduces the background of non-recombinant vector in the library.
  3. Ligate 180 µg vector with ~20 µg insert DNA as described in LibraryConstruct.doc, Step 1.
  4. Process the ligation mixture, electroporate and propagate the library as described in LibraryConstruct.doc, Steps 6-12.

REFERENCES

1.Folgori, A., Tafi, R., Meola, A., Felici, F., Galfre, G., Cortese, R., Monaci, P., and Nicosia, A. (1994) EMBO Journal13, 2236-2243.

2.Felici, F., Luzzago, A., Folgori, A., and Cortese, R. (1993) Gene128, 21-27.

3.Prezzi, C., Nuzzo, M., Meola, A., Delmastro, P., Galfre, G., Cortese, R., Nicosia, A., and Monaci, P. (1996) Journal of Immunology156, 4504-4513.

4.Luzzago, A., Felici, F., Tramontano, A., Pessi, A., and Cortese, R. (1993) Gene128, 51-7.

5.Steward, M. W., Stanley, C. M., and Obeid, O. E. (1995) J Virol69, 7668-73.

6.Geysen, H. M., Rodda, S. J., and Mason, T. J. (1986) Molecular Immunology23, 709-15.

7.Davies, D. R., Padlan, E. A., and Sheriff, S. (1990) Annu Rev Biochem59, 439-73.

8.Berzofsky, J. A., Buckenmeyer, G. K., and Hicks, G. (1982) J Immunol128, 737-41.

9.Schulze-gahmen (1986) Eur j biochem159, 283.

10.Van Regenmortel, M. H. V. (1989) Immunology Today10, 266-271.

11.Kaumaya, P. T., VanBuskirk, A. M., Goldberg, E., and Pierce, S. K. (1992) J Biol Chem267, 6338-46.

12.Zhong, G., Smith, G. P., Berry, J., and Brunham, R. C. (1994) Journal of Biological Chemistry269, 24183-8.

13.Smith, G. P., and Petrenko, V. A. (1997) Chemical Reviews97, 391-410.

14.Keller, P. M., Arnold, B. A., Shaw, A. R., Tolman, R. L., Van Middlesworth, F., Bondy, S., Rusiecki, V. K., Koenig, S., Zolla-Pazner, S., Conard, P., and et al. (1993) Virology193, 709-16.

15.Lundin, K., Samuelsson, A., Jansson, M., Hinkula, J., Wahren, B., Wigzell, H., and Persson, M. A. (1996) Immunology89, 579-86.

16.Motti, C., Nuzzo, M., Meola, A., Galfre, G., Felici, F., Cortese, R., Nicosia, A., and Monaci, P. (1994) Gene146, 191-8.

17.Stoute, J. A., Ballou, W. R., Kolodny, N., Deal, C. D., Wirtz, R. A., and Lindler, L. E. (1995) Infection & Immunity63, 934-9.

18.Orlandi, R., Menard, S., Colnaghi, M. I., Boyer, C. M., and Felici, F. (1994) European Journal of Immunology24, 2868-2873.

19.Chargelegue, D., Obeid, O. E., Shaw, D. M., Denbury, A. N., Hobby, P., Hsu, S. C., and Steward, M. W. (1997) Immunol Lett57, 15-7.

20.Demangel, C., Lafaye, P., and Mazie, J. C. (1996) Mol Immunol33, 909-16.

21.Meola, A., Delmastro, P., Monaci, P., Luzzago, A., Nicosia, A., Felici, F., Cortese, R., and Galfre, G. (1995) Journal of Immunology154, 3162-3172.

22.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning : a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

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