Construction of Large Naive Fab Library

Antibody Phage Display Methods and Protocols
Edited by
Philippa M. O’Brien
and
Robert Aitken
University of Glasgow, Glasgow, Scotland, UK, 2002

Construction of Large Naïve Fab Libraries Hans J. W. de Haard, p87

2. Materials
1. Ficoll-Paque(researchgrade)(AmershamPharmaciaBiotech,Uppsala,Sweden).
2. BufferA:4Mguanidineisothiocyanate,25mMcitricacid,0.5%(w/v)N-lauroyl sarcosine, 1% (v/v) 2-mercaptoethanol, pH 7.0.
3. Ultra-turraxT25(Janke&Kunkel,Steufen,Germany).
4. 2MSodiumacetate(NaAC),pH4.0.
5. H2O-saturatedphenol.
6. Chloroformisoamylalcohol(24 1).
7. Absolute ethanol.
8. Randomhexamerprimers(AmershamPharmaciaBiotech).
9. Deoxyribonucleoside triphosphates (dNTP) (deoxynucleotides, sequencing- grade solutions) (Amersham Pharmacia Biotech).
10. Dithiothreitol(100mM).
11. RNasinRibonucleaseInhibitor(Promega,Madison,WI).
12. Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and reaction buffer (Life Technologies, Grand Island, NY).
13. AmpliTaq Gold (Perkin-Elmer/Roche, Branchburg, NJ).
14. QIAex-IIextractionkit(Qiagen,Hilden,Germany).
15. SfiI,BstEII,NotI,ApaLI,AscI(NewEnglandBiolabs,Beverly,MA).
16. Microcon-50(Amicon/Millipore,Bedford,MA).
17. QiagenPlasmidMegakit(Qiagen)orNucleobondAX-500andAX-2000plasmid purification kit (Clontech, Palo Alto, CA).
18. T4DNAligase(Promega).
19. Genepulserandpulsecontroller;electroporationcuvets(0.2-cmgap)(Bio-Rad, Hercules, CA).
20. LuriaBertani(LB)solidandliquidmedia.Refertoindexforcomposition.
21. Autoclave a solution of 16 g/L Bacto-tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0. After cooling, add filter-sterilized glucose (GLU) solution to a final concentration of 2% (2TY–GLU). For 2TY, omit the glucose. For solid media, add agar at 15 g/L prior to autoclaving.
22. Kanamycin.
23. Phageprecipitant:20%polyethylene6000,2.5MNaClinH2O.

3. Methods
3.1. RNA Isolation (see Note 1)
1. Peripheral blood lymphocytes purified by centrifugation on Ficoll-Paque gradi- ents can form one source of starting material (see Note 2). Recover the layers containing lymphocytes, pellet the cells, and wash with phosphate-buffered saline (PBS) (see Note 3). Lymphocytes residing in bone can be obtained by flushing a segment with PBS using a syringe. Dissolve the cell pellets from 1 L blood in 30 mL buffer A. Shear chromosomal DNA by passing the suspension several times through a syringe fitted with a narrow needle.
2. Solid tissues, such as spleen or lymph nodes, are alternative starting materials (see Note 2). Cut 0.5–1.0 g of the sample into small pieces. After addition of 30 mL buffer A, homogenize the tissue rapidly with an Ultra-turrax homogenizer and a potter. Remove debris by centrifugation (10 min at 5000g) and pass the supernatant through a narrow syringe to shear chromosomal DNA.
3. After adding 0.1 vol 2 M NaAC, pH 4.0, extract the mixture with an equal volume of H2O-saturated phenol and 1/2 vol chloroform–isoamyl alcohol (24 1). Incubate the mixture on ice for 15 min to improve phase separation. Following centrifugation (10 min at 5000g), transfer the H2O phase to a new tube and re-extract with phenol and chloroform.
4. PrecipitatetheRNAwith0.75volethanolduring16hat–20°C.Pelletthenucleic acids (30 min at 13,000g), dissolve in 0.1 vol autoclaved H2O, and precipitate again by adding 0.01 vol 2 M NaAC, pH 4.0 and 0.25 vol ethanol. Store the RNA under ethanol at –20 or –80°C until required.
5. Quantify the RNA by measuring the optical density at 260 nm. Typically, 1.0–1.5 × 109 peripheral blood lymphocytes (with approx 10–20% B cells) can be isolated from 1 L blood, which yields 1.2–2.5 mg total RNA, when using the method described. One-half gram spleen yields 1.2 mg and 0.5 g lymph node yields 1.5 mg total RNA (see Note 4).
3.2. Amplification of Variable-Region Genes
1. Prepare random primed cDNA from 250 μg total RNA. Centrifuge an appropri- ate volume of ethanol mixture (10 min at 13,000g), and, after dissolving in autoclaved H2O, heat-denature the RNA for 5 min at 65°C in the presence of 20 μg random primers in a total vol of 50–100 μL (see Note 5).
2. Place the reaction vessel on ice and add RT buffer, dithiothreitol (to 10 mM), dNTP (to 250 μM), RNasin (800 U) and MMLV-RT (2000 U), yielding a total volume of 500 μL. Incubate for 2 h at 42°C, then terminate the reaction by a phenol–chloroform extraction (see Note 6). Precipitate the cDNA from the aqueous layer by adding 0.1 vol NaAC and 2 vol ethanol, and centrifuge (10 min at 13,000g). Wash the pellet (70% ethanol), centrifuge, and dissolve the dried pellet in 85 μL H2O. Use the cDNA solution immediately, or store at –20°C.
3. Amplify the human variable-region genes by PCR with the oligonucleotides described in Table 1 (see Note 7). IgM-derived heavy-chain (HC) variable regions are obtained by a primary PCR, with an IgM constant-region primer combined with separate VH-family-specific Back primers, which anneal to the 5  end of the V regions.   and   LC-derived variable regions are amplified with a set of C -For and C -For primers annealing to the 3  end of the constant domain and separate V - and V -family-specific Back primers. Perform PCR in a volume of 50 μL using AmpliTaq Gold polymerase using the supplier’s buffer, 0.2 mM dNTPs, 500 nM of each primer, and 2.5 μL cDNA as template. After activation of the polymerase by heating at 94°C for 11 min, carry out 28 cycles of amplification (30 s denaturation at 94°C, 30 s annealing at 55°C, and 2.5 min elongation at 72°C) (see Note 8). Nine separate reactions generate the different VH-family- derived amplicons; for the LC families, six separate V C  products and 11 V C  products (C 2 and C 7 primers combined in one reaction) are generated.
4. Purify the PCR products from 1.5% agarose gel with a QIAex-II extraction kit, and elute in 40 μL H2O (see Note 9). The VH regions are reamplified from individual HC-derived amplicons using the corresponding SfiI-tagged VH-Back and a set of JH-For primers, which contain a BstEII site (see Notes 10 and 12). The complete V C  and V C  products are reamplified with ApaLI- and AscI- tagged primers. As input to these reactions, use 100–200 ng purified product in 100 μL PCR reactions (see Note 8). Sufficient DNA for cloning should be obtained from 25 cycles (see Subheading 3.2, step 3).

3.3. Construction of Primary VH, V , and V  Libraries
1. Purify the PCR products appended with restriction sites from 1.5% agarose gels (see Note 9).
2. Pool equal amounts of DNA from the different VH families and digest with SfiI and BstEII at 50°C. Similarly, V C  and V C  fragments (family-derived PCR products pooled, but   and   LCs kept separately) are digested with ApaLI and AscI at 37°C. Run all digests for 16 h with 50–100-fold excess of enzyme (U vs μg DNA) (see Note 10).
3. Remove restriction enzymes and salts by spin-dialysis against H2O using a Microcon-50 unit. Determine the amount of DNA recovered on an agarose gel.
4. Purify the phagemid vector (e.g., pCES1: map and sequence shown in Fig. 1) from 1 L cultures using Nucleobond AX-500 or AX-2000 kits or a Qiagen plasmid mega kit. Digest approx 400 μg DNA with SfiI and BstEII or with ApaLI and AscI and purify from 1% agarose gel, using the QIAex-II kit (see Notes 11 and 12).
5. Determine the optimal ratio of vector fragment with test ligations. Ligate 25 ng vector with three different amounts of fragment, varying from 2 to 25 ng, in a volume of 20 μL using 1 U T4 DNA ligase and the buffer supplied by the manufacturer. After incubation for 15–60 min at room temperature, use 3 μL ligation reaction for transformation of 40 μL electrocompetent TG1 cells (see Note 13) with 0.2-cm cuvets (2.5 kV pulse at 25 μF and 200  ). Immediately after electroporation, add 1 mL 2TY–GLU, and plate 100 and 1 μL onto LB plates containing 100 μg/mL ampicillin and 2% glucose LB medium (LB–AMP–GLU). Compare the number of transformants with those found on the control (ligation of vector alone) to reveal which ratio is to be used (see Notes 11 and 12).
6. For the library construction, ligate 1–5 μg vector with the optimal amount of fragment (varying from 0.1 to 1.5 μg) for 16 h at room temperature in a total volume of 100–200 μL using 9 U T4 DNA ligase. Desalt the reaction mixture by spin-dialysis against H2O with a Microcon-50 unit.
7. Divide the ligation mixture into 20–40-μL aliquots and separately electroporate with 100–150 μL freshly prepared TG1 cells (see Notes 13 and 14). Immediately after transformation, transfer the cells in 1 mL 2TY–GLU medium to a tube. Pool the remaining fractions belonging to the same ligation and rinse the cuvets with 1 mL medium. To establish the library size, prepare a dilution series (10–3, 10–4, 10–5, and 10–6) in medium and plate 100 μL onto small (9 cm diameter) LB–AMP–GLU plates. Plate the rest of the transformation mixture on large LB–AMP–GLU plates (24 × 24 cm) to allow outgrowth of individual clones (approx 108–109 clones/plate) (see Notes 15 and 16). Incubate overnight at 37°C.
8. Scrape plates with 2TY–AMP–GLU medium to get suspensions with an OD600, of 50 to 100. Add glycerol to a final concentration of 20%, measure the OD600 and store in aliquots at –80°C as individual primary library stocks (see Note 17).
3.4. Combining VH, V , and V  into Fab Single-Pot Library
1. Thawglycerolstocksfromtheindividualprimarylibrariesandinoculateasample containing at least 10-fold more cells than the library size to 1 L LB–AMP–GLU liquid medium. After 8–16 h of growth, purify plasmid DNA using the previously mentioned kits (see Subheading 3.3., step 4).
2. Digest plasmid DNA (approx 500 μg) from the LC and HC repertoires with SfiI and BstEII (or NotI) (see Note 12). Purify vector from the LC repertoires from 1% agarose gel, and VH fragments from the HC repertoires using 1.5% agarose gels (see Subheading 3.2., step 4).
3. Perform test ligations and electroporations to establish the optimal ratios of vector fragment (see Subheading 3.3., step 5).
4. Upscale the ligation reactions and electroporation of freshly prepared TG1 cells to yield the final library (see Subheading 3.3., steps 6 and 7, and Notes 13–15). Store individual combinations of   and   LC-derived libraries with the VH fragments separately as glycerol stocks (see Subheading 3.3., step 8, and Notes 16 and 17).
3.5. Preparation of Phage
1. Prepare helper phage by infecting log-phase TG1 bacteria with M13K07 or VCSM13 phage at different dilutions for 30 min at 37°C and plating out in top agar onto 2TY plates.
2. Take phage from a small plaque, and resuspend in 3 mL liquid 2TY medium. Add 30 μL overnight culture of TG1 and grow for 2 h at 37°C.
3. Dilute the culture in 1 L 2TY medium and grow for 1 h. Add kanamycin to 50 μg/mL and grow for 16 h at 37°C.
4. Removecellsbycentrifugation(10minat5000g)andprecipitatephagefromthe supernatant by addition of 0.25 vol of phage precipitant. After 30 min incubation on ice, collect the phage particles by centrifugation during 10 min at 5000g. Resuspend the pellet in 5 mL PBS and sterilize through a 0.22-μm filter.
5. Titratethehelperphagebydeterminingthenumberofplaque-formingunits(pfu) on 2TY plates with top-agar layers containing 100 μL TG1 (saturated culture) and dilutions of phage. Dilute the phage stock solution to 1 × 1013 pfu/mL and store in small aliquots at –20°C.
6. BeforepreparationofphagefromtheFablibrary,prepareamasterstocksolution from the individual glycerol stocks by combining appropriate volumes (according to the size of each library) into a single mixture. Using a sample large enough to ensure the presence of at least 10 bacteria from each clone present in the final library, inoculate an appropriate volume of 2TY–AMP–GLU medium to obtain a log phase culture (see Note 18).
7. Growforafewhoursat37°CuntiltheculturereachesanOD600of0.5–0.9.Take a sample containing at least that number of bacteria at least 10-fold the size of the library. Add helper phage at a multiplicity of infection of 10–20 (i.e., the number of phage particles/host cell). Infect cells by incubating the suspension during 30 min at 37°C without shaking and an additional period of 30 min with shaking. Collect infected cells by centrifugation (10 min at 5000g) and resuspend in 2TY medium containing 100 μg/mL ampicilin and 25 μg/mL kanamycin. Grow the culture during 16 h at 30°C (see Note 15).
8. Precipitate phage particles from the supernatant as described before (step 4). Resuspend the phage pellet in 0.05 vol (i.e., 50 mL/L culture) of PBS and remove cellular debris by centrifugation (10 min at 5000g). To remove Ab fragments not associated to phage particles, carry out a second polyethylene glycol precipitation. Resuspend the phage pellet in 0.005 vol of PBS, clarify again by centrifugation, and pass through a 0.45-μm filter. Add an equal volume of glycerol and store the phage suspension at –80°C.
9. Titrate the phage as the number of transducing units by infecting TG1 cells and counting the clones after plating on LB–AMP–GLU plates. Adjust the phage stock solution with 50% glycerol/PBS, to a final titer of 1013 transducing U/mL. Divide the phage solution into 1-mL aliquots and store at –80°C. For each selection, thaw one tube. Remove glycerol by polyethylene glycol precipitation and resuspend the phage pellet in 1 mL buffer solution compatible with the desired selection procedure (see Note 19).
4. Notes
1. The method for RNA isolation (7) is robust and relies on the inactivation of RNases by the combined action of the chaotropic agent, guanidine isothiocya- nate and 2-mercaptoethanol. Wear gloves during the isolation and use freshly autoclaved H2O and disposables.
2. Process tissues and blood samples as soon as these are taken from the donor, since prolonged storage on ice or at 4°C results in the isolation of degraded RNA. Freezing tissues rapidly in liquid nitrogen and storage at –80°C will also yield RNA of poor quality.
3. Thetotalnumberofperipheralbloodlymphocytescanbedeterminedbycounting a sample of cells diluted in Turks solution on a Bürker-Turk cell.
4. ThequalityoftheRNApreparationshouldbecheckedonanappropriategel(for instance, on systems using glyoxal- or formaldehyde-denatured RNA) (see Fig. 2), before starting cDNA synthesis. This analysis is more useful than determining the ratio between OD260 and OD280, which reveals the presence of protein; however, this will not interfere with cDNA synthesis.
5. Instead of total RNA, poly A+-containing transcripts purified with oligo(dT) beads can be used for synthesis of random primed cDNA. Random primers are preferred, instead of Ig-derived oligonucleotide primers, to generate cDNA, because the latter will yield nonspecific bands during PCR. Abundant amounts of total RNA in cDNA preparations can affect the efficiency of amplification. This can be solved by hydrolysis of RNA with NaOH after cDNA synthesis.
6. Itisnotstrictlynecessarytophenol-extractandprecipitatecDNA.Samplesfrom the RT mixture can be used directly as template for PCR.
7. All primary PCRs should be carried out with separate Back primers to amplify even rarely occurring V genes.
8. Large amounts of cDNA and a limited number of cycles are used during PCR to obtain maximal diversity and to prevent the overamplification of just a few V regions. As a control, 1-μL 50-fold-diluted cDNA solution can be added as template in separate reactions, which should yield products, using the described PCR protocol. For the same reason, a large input of gel-purified product is used during reamplification with the tagged primers. Yields can be checked by analysis of 4 μL unamplified PCR mixture on a gel.
9. EfficientQIAexpurificationofthepooledVH,V -,andV -derivedampliconsor fragments digested from library-derived plasmid DNA is achieved by dissolving