Editing Human Phage Display Library

2. Materials<br/>1. Fresh source of lymphoid tissue from which mRNA can be isolated.<br/>2. Kit for the preparation of mRNA (e.g., an oligo(dT)-purification system, such as the Quickprep mRNA Kit; Amersham Pharmacia Biotech).<br/>3. Kit for the synthesis of cDNA (e.g., “First-strand cDNA synthesis kit”; Amersham Pharmacia Biotech).<br/>4. PCR reagents: Taq DNA polymerase with 10X Taq DNA polymerase buffer (Boehringer Mannheim), a stock of deoxyribonucleoside triphosphates (dNTPs) (5 mM each) (Pharmacia), PCR H2O [ACS] reagent; Aldrich cat. no. 32, 007-2).<br/>5. Oligonucleotide primers at 10 μM. Tables 1–4 show primer sets currently used in the author’s laboratory for the construction of human scFv libraries.<br/>6. Buffer-saturated phenol UltraPure™ (Gibco-BRL).<br/>7. Chloroform.<br/>8. Absolute ethanol (–20°C); 70% (v/v) ethanol (–20°C).<br/>9. Kit for the isolation of DNA from gels (e.g., GeneClean, Bio101).<br/>10. H2O (ACS reagent grade).<br/>11. Phage display vector(s) and appropriate restriction enzymes for cloning. In the example described, the vectors, pCANTAB6 and pCANTAB3his6, are used with the enzymes Sfi I, NotI, XhoI, HindIII, ApaLI, although the principles of library construction are not limited to these vectors or restriction enzymes. Samples of pCANTAB6 and pCANTAB3his6 can be obtained from Cambridge Antibody Technology under a standard materials transfer agreement.<br/>12. Kit for medium-scale isolation of plasmid DNA (e.g., Qiagen midipreps).<br/>13. Ligation kit (e.g., “Rapid ligation kit,” Amersham Pharmacia Biotech, cat. no. RPN 1507), 5X ligation buffer (500 mM Tris-HCl, 25 mM MgCl2, pH7.4).<br/>14. Electrocompetent Escherichia coli TG1.<br/>15. Bio-Rad Gene Pulser™ and suitable electroporation cuvets.<br/>16. 2TY as liquid and solid media (refer to index or ref. 8 for composition).<br/>17. Glucose 20% (w/v) sterile-filtered.<br/>18. Ampicillin (100 mg/mL stock) and kanamycin (50 mg/mL stock), both filtersterilized.<br/>19. Kit for the purification of PCR products (e.g., Wizard PCR preps, Promega).<br/>20. 20% (w/v) Polyethylene glycol (PEG) 8000, 2.5 M NaCl.<br/>21. TE buffer (10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid, pH 8.0).<br/>22. Ultrapure cesium chloride (CsCl).

<br/>3.1. Synthesis of Primary cDNA Template (see Note 1)

1. Isolate approx 1 × 107 cells peripheral blood lymphocytes from 50 mL whole blood (6) (see Note 2).<br/>2. Immediately isolate the mRNA from the cell pellet (7) using an oligo(dT)-purification system, following the manufacturer’s instructions.<br/>3. Synthesize first-strand cDNA from the mRNA template with random hexamersusing a kit. Follow the manufacturer’s instructions (3).

<br/>3.2. VH Repertoire Construction<br/>1. Perform separate 50 μL PCR reactions for each VH Back primer using the following cycling parameters: 94°C 1 min, 55°C 1 min, 72°C 2 min for 30 cycles; final extension at 72°C for 10 min (see Note 3). Each reaction comprises 5.0 μL 10X Taq buffer, 2.5 μL 5 mM dNTP stock, 2.5 μL individual VH BackSfiI primer at 10 μM, 2.5 μL equimolar mix of JH1-6ForXhoI primers at 10 μM total, 5.0 μL first-strand cDNA mix (typically 0.5 ng cDNA), 31.5 μL PCR H2O, and 1.0 μL Taq polymerase (5 U).<br/>2. Pool the PCR reactions and concentrate the DNA by phenol/chloroform extraction, followed by ethanol precipitation (8). Resuspend the DNA pellet in ACS reagent-grade H2O.<br/>3. Digest the DNA with SfiI using the reaction buffer provided by the distributor, noting that the optimal temperature for this enzyme is 50°C. Extract with phenol/chloroform and concentrate the DNA by ethanol precipitation.<br/>4. Digest the products with XhoI. After digestion, heat-inactivate the enzyme at 65°C for 20 min.<br/>5. Run the products on a TAE agarose gel, and check that the VH segments are approx 350 bp in size.<br/>6. Excise the band and purify the VH fragments using a commercial kit, following the manufacturer’s recommended protocol. Estimate the concentration of the VH product by comparison with a DNA marker (typically 􀁨 DNA digested with HindIII) of known concentration on a 1% (w/v) TAE agarose gel.<br/>7. Digest and concentrate the pCANTAB6 vector (see Note 4) with SfiI and XhoI restriction enzymes (steps 2 and 3 above). Estimate the concentration of recovered DNA by comparison with known DNA markers (step 5).<br/>8. Set up a series of trial ligations covering a range of molar ratios of insert:vector (e.g., 1:1, 2:1, 4:1, 1:2) and a fixed concentration of pCANTAB6 within the range specified by the manufacturer of the ligation kit (typically, 50–500 ng vector DNA) (see Note 5). Carry out the ligation reaction according to the manufacturer’s instructions.<br/>9. Precipitate the DNA with ethanol (8), redissolve in the smallest-possible volume of ACS reagent-grade H2O, and electroporate into E. coli TG1 (2.5 V, 200 􀀱) (see Note 5). Add 2TY containing 2% glucose (2TYG) and allow the cells to recover for 1 h with shaking at 200 rpm in a 37°C incubator.<br/>10. Plate aliquots from each transformation to 2YTG plates containing ampicillin (100 μg/mL; 2TYAG) and incubate overnight at 30°C. Confirm that background levels of vector self-ligation are minimal and which ligation ratio yields the highest numbers of transformants.<br/>11. From the planned size of the VH library (see Note 6) and the recovery of transformants from the optimal ligation, calculate the number of ligation reactions required for library construction. Set up ligations and precipitate the products as before (steps 6 and 7).<br/>12. Perform at least 40 electroporations with electrocompetent E. coli TG1 cells (step 7), and let the cells recover for 1 h in 2TYG with shaking at 200 rpm at 37°C. Pool the cells from all electroporations, and take a small aliquot for plating at 10-fold serial dilutions onto 2TYAG plates to determine the size of the library.<br/>13. Centrifuge the rest of the cells at 1200g for 10 min, remove the supernatant, and resuspend in 1–2 mL 2TY. Plate out the total library on four large 243 × 243 mm 2TYAG plates.<br/>14. Incubate all plates overnight at 30°C. Determine the size of the library from the titer plates: this should be in the region of 1 × 107 to 1 × 108 individual recombinants. Scrape cells from the large plates and prepare glycerol stocks for storage at –70°C (see Subheading 3.4., step 7).

3.3. VL Repertoire Construction (see Note 7)<br/>1. Perform 50 μL PCR reactions using the same cycling parameters as outlined for recovery of VH products (see Subheading 3.2.). Use 5 μL first-strand cDNA mix as the template with the following primer combinations: V􀁨BackApaLI + J􀁨1–5ForNotI primer mix and VκBackApaLI + Jκ 1–5ForNotI primer mix.<br/>2. Pool and concentrate the PCR products as described previously (see Subheading&nbsp; 3.2.), and digest with ApaLI. Extract with phenol/chloroform, precipitate the DNA, then digest with NotI. Heat-inactivate at 65°C for 20 min and gel-purify the VL&nbsp; fragments. Estimate the concentration of purified insert as described previously (see Subheading 3.2.). The size of the resulting VL fragments will be 350 bp.<br/>3. Digest the pCANTAB3his6 vector sequentially with ApaLI and NotI restriction enzymes (step 2) and concentrate the cut vector by performing a phenol/ chloroform extraction, followed by ethanol precipitation. Estimate the concentration of DNA recovered by comparison with markers.<br/>4. Perform ligation reactions and subsequent electroporations as described for the VH repertoire (see Subheading 3.2.). The size of the Vκ and V􀁨 repertoires should&nbsp; be between 1 × 105 and 1 × 106 individual recombinants.<br/>5. To introduce the (Gly4Ser)3 scFv-linker into the finished VL repertoire, PCRamplify the linker from an existing scFv, together with an irrelevant (dummy) VH fragment. First, pick a single colony of an irrelevant clone that possesses the required scFv linker and PCR-amplify with primers pUC19rev and fdtetseq primers (see Table 3) using the cycling parameters described previously (see Subheading 3.2.).<br/>6. Assuming that gel analysis shows a single product, isolate the amplicon using a commercial kit and digest with ApaLI. Phenol/chloroform extract, ethanolprecipitate, and digest with HindIII to generate a product of ~450 bp. Heatinactivate (65°C for 20 min), purify the DNA from a 1% TAE agarose gel, and estimate its concentration by comparison with DNA markers.<br/>7. Prepare the pCANTAB3his6 vector containing the VL repertoire on midiprep scale and sequentially digest 10 μg with ApaLI and HindIII restriction enzymes (step 6).

8. Concentrate the digested VL repertoire by phenol/chloroform extraction, followed by ethanol precipitation. Gel-purify the large DNA fragment and estimate its<br/>concentration by comparison with markers.<br/>9. Perform sufficient ligation reactions to ligate approx 0.4 μg dummy VH-linker DNA fragment into a 1-μg pool of the Vκ and V􀁨 libraries.<br/>10. Electroporate into E. coli TG1 cells, and plate out as described previously (see Subheading 3.2.). Aim to generate between 1 × 106 and 1 × 107 recombinants,<br/>carrying VL inserts with upstream scFv linker and dummy VH.&nbsp;

3.4. Construction of the scFv Library (see Notes 8 and 9)<br/>&nbsp;1. Amplify the VH and linker-VL DNA fragments separately from each of the cloned repertoires. Perform 50 μL PCR reactions using the cycling parameters described<br/>previously (see Subheading 3.2.), amplifying the VH repertoire with pUC19rev and JHFor primers and the VL repertoire with reverse JH and fdtetseq primers.<br/>Purify the products from 1% TAE agarose gels and estimate DNA concentrations&nbsp; by comparison with markers.<br/>2. Combine equal amounts of the VH and linker-VL PCR products (5–20 ng each), increase the total volume to 100 μL with ACS, reagent-grade H2O prior<br/>to recovery of the DNA by ethanol precipitation. Resuspend the DNA pellet in 25 μL H2O.<br/>3. To perform the assembly reaction, add the following reagents to the poo led VH and linker-VL products, and perform 25 cycles of 94°C for 1 min, followed by<br/>65°C for 4 min: 3.0 μL 10X Taq buffer, 1.5 μL 5 mM dNTP stock, and 0.5 μL Taq polymerase (2.5 U).<br/>4. Prepare 50 μL pull-through PCR reactions, pairing each VH BackSfiI primer with either the Jκ1-5ForNotI primer mix or the J􀁨1-5ForNotI primer mix. Replicates of each reaction are advisable, to maximize the diversity of the final library. Using 5.0 μL assembly DNA/reaction, amplify with cycling parameters described previously (see Subheading 3.2.). The correct size of the assembled construct is around 700 bp.<br/>5. Pool and concentrate the PCR products by phenol/chloroform extraction, followed by ethanol precipitation. Sequentially digest with SfiI and NotI restriction endonucleases as described previously (see Subheadings 3.2. and 3.3.).<br/>6. Gel-purify the digested scFv assembly construct and ligate with SfiI/NotI digested<br/>pCANTAB6 after determining the optimum insert:vector ratio as described<br/>previously (see Subheading 3.2.). Perform at least 100 electroporations, pool<br/>into batches, and plate out each batch on large 243 × 243 mm 2TYAG plates.<br/>Determine the total size of the library by taking aliquots from each batch and<br/>plating out serial dilutions on 2TYAG. The final library should contain in the<br/>region of 1 × 108 to 1 × 109 individual recombinants. Clones picked from these<br/>plates can be used to characterize the library (see Note 9).<br/>7. Scrape the large plates, using 5 mL 2TY/plate, and pool the cells in 50-mL Falcon<br/>tubes. Add 0.5 vol 50% (v/v) glycerol to each tube, and ensure homogeneous resuspension of the cells by mixing on a rotating wheel for 30 min. Determine<br/>cell density by optical density measurement at 600 nm. Store the library in<br/>aliquots at –70°C.<br/>3.5. Preparation of Library Phage (see Note 10)<br/>1. Inoculate 500 mL 2TYG with 1010 cells from the library glycerol stock and<br/>incubate at 37°C with shaking at 250 rpm until the optical density at 600 nm<br/>reaches 0.5–1.0.<br/>2. Add M13KO7 helper phage to a final concentration of 5 × 109 pfu/mL, and<br/>incubate for 30 min at 37°C without shaking, then for 30 min with gentle shaking<br/>(200 rpm), to allow phage infection.<br/>3. Recover the cells by centrifugation at 2200g for 15 min and resuspend the pellet<br/>in the same volume of 2TYAK (2TY containing 100 μg/mL ampicillin, 50 μg/mL<br/>kanamycin). Incubate overnight at 30°C with rapid shaking (300 rpm).<br/>4. Pellet the cells by centrifugation at 7000g for 15 min at 4°C and recover the<br/>supernatant containing the phage into prechilled 1-L bottles.<br/>5. Add 0.3 vol of PEG/NaCl. Mix gently and allow the phage to precipitate for<br/>1 h on ice.<br/>6. Pellet the phage by twice centrifuging at 7000g for 15 min in the same bottle at<br/>4°C. Remove as much of the supernatant as possible and resuspend the pellet<br/>in 8 mL TE buffer.<br/>7. Recentrifuge the phage in smaller tubes at 12,000g for 10 min and recover the<br/>supernatant, which will now contain the phage. Ensure that any bacterial pellet<br/>that appears is left undisturbed.<br/>8. Add 3.6 g of caesium chloride to the phage suspension and raise the total volume<br/>to 9 mL with TE buffer. Using an ultracentrifuge, spin the samples at 110,000g,<br/>23°C, for at least 24 h.<br/>9. After ultracentrifugation, the phage should be visible as a tight band, which<br/>can be recovered by puncturing the tube with a 19-gage needle plus syringe<br/>and careful extraction.<br/>10. Dialyze the phage against two changes of 1 L TE at 4°C for 24 h.<br/>11. Finally, titer phage stocks by infecting TG1 cells with dilutions of phage stock,<br/>plating to 2TYAG, incubation, and enumeration of the numbers of ampicillinresistant<br/>colonies that appear. The phage can then be stored in aliquots at 4°C for<br/>long periods (see Note 10), ready for screening (see Note 11).<br/>4. Notes<br/>1. Rapid processing of fresh tissue samples is essential if the full diversity of the<br/>Ab repertoire is to be recovered. If some loss of diversity is acceptable (perhaps<br/>when preparing libraries from infected or immunized individuals, rather than<br/>in developing a comprehensive naïve library) tissue, mRNA, or cDNA product<br/>can be stored at –70°C.<br/>2. RNA isolation (7) from Ficoll-isolated leukocytes, as described by Marks et<br/>al. (6), is the method of choice. 50 mL of blood should yield approx 1 × 107<br/>cells, which in turn yield about 10 μg total RNA, of which 1–5% is mRNA. It<br/>is important to ensure that there is enough cDNA for all the VH and VL PCR<br/>reactions planned, each of which requires 0.5 ng cDNA.<br/>3. The PCR primers employed are based on those published by Marks et al. (6),<br/>and/or gene sequences in the V-BASE directory. The 5􀁶 and 3􀁶 VH primers include<br/>SfiI and XhoI restriction sites, respectively, to allow for cloning (see Table 1).<br/>Include “no template” controls and check all PCR products on 1–2% (w/v)<br/>TAE agarose gels to ensure that a clean product of the expected size has been<br/>generated.<br/>4. Plasmid DNA (pCANTAB6 or pCANTAB3his6) is prepared either by the alkali<br/>lysis method (and subsequently caesium-banded as detailed in Sambrook<br/>et al. [8]), or by using a commercial kit (medium-scale). Approximately 20 μg<br/>Cs-banded vector will yield ~5–10 μg purified cut vector. Efficient digestion with<br/>both enzymes is crucial to avoid self-ligation of the vector and high backgrounds<br/>at transformation.<br/>5. A “vector only” ligation control should be included to determine the background<br/>caused by nonrecombinants. Protocols for the preparation of electrocompetent<br/>E. coli TG1 cells and subsequent electroporations are described in Sambrook<br/>et al. (8) and by other contributors to this volume.<br/>6. In most cases, a repertoire of ~1 × 107–1 × 108 recombinants can be generated if<br/>0.5 μg digested VH segments are ligated with 1.5 μg digested vector.<br/>7. VL κ and VL 􀁨 gene fragments are amplified separately using each back primer<br/>in combination with the appropriate equimolar mixture of the Jκ or J􀁨 Forward<br/>primers (see Table 2). After recovery of the combined VL repertoire, the next<br/>stage is to clone in the (Gly4Ser)3 scFv linker from an existing scFv, together<br/>with a dummy VH, recovered by PCR from an irrelevant clone. Primer sequences<br/>are shown in Table 3.<br/>8. Final scFv library construction involves the amplification of VH and linker-VL<br/>DNA fragments from each cloned repertoire (VH in pCANTAB6 and linker-VL<br/>in pCANTAB3his6), followed by assembly on the JH region and amplification<br/>by pull-through PCR (see Table 3 for pull-through PCR primers). The resulting<br/>scFv constructs (VH-linker-VL) are digested with SfiI and NotI and ligated into<br/>SfiI/NotI digested pCANTAB6.<br/>9. Quality control analysis of the library is routinely performed by two methods to<br/>determine the percentage of recombinant clones and the level of library diversity.<br/>For both methods, the first stage is to PCR-amplify the scFv insert from 50<br/>randomly picked clones/repertoire, using the vector primers, pUC19 reverse and<br/>fdtetseq, as described in Subheading 3.2. Digestion of the PCR products with<br/>BstNI restriction endonuclease and agarose gel electrophoresis can then be used<br/>to visualize the restriction profile for each clone. The low cost and technical<br/>simplicity of this approach are its main strengths, but, as a means to assess the<br/>diversity of a library, it is limited by the resolving power of the agarose gel. Greater resolution and sensitivity can be achieved with polyacrylamide gels<br/>and silver staining (8), but sequence analysis with fluorescent dideoxy chain<br/>terminators directly from the PCR products is clearly a better method, since it is<br/>sensitive to single-base differences between clones beyond the BstNI recognition<br/>sequence. Each clone picked should carry a unique combination of VH and VL<br/>sequences.<br/>10. The resultant phage are purified by PEG precipitation and caesium-banding, and,<br/>as a result, are stable at 4°C for 2 yr. Phage prepared by PEG precipitation alone<br/>should only be stored at 4°C for 1–2 wk.<br/>11. The affinities of Abs directly isolated from scFv repertoires constructed in this<br/>manner without further engineering can be in the subnanomolar range and tend to<br/>have slower off-rates than those derived from rodent immune responses, smaller<br/>scFv repertoires, or large synthetic Fab libraries (3).

Primers
<pre>pCMVFor
CGCAAATGGGCGGTAGGcGTG
pCMVrev
CAACATAGTTAAGAATACCAGTC

VHH1SfiFor     ____ SfiI____Nco1
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG
           L  A  A  Q  P  A  M  A  Q  V  Q  L  V  Q  S    
VHH2SfiFor     ____ SfiI____ VH
CATGCCATGACTCGCGGCCCAGCCGGCC ATGGCCCAGGTCACCTTGAAGGAGTCTGG
           L  A  A  Q  P  A   M  A  Q  V  T  L  K  E  S    


VHH3SfiFor     ____ SfiI____
CATGCCATGACTCGCGGCCCAGCCGGCC ATGGCCCAGGTGCAGCTGGTGGAGTCTGG
  M  P  -  L  A  A  Q  P  A   M  A  Q  V  Q  L  V  E  S   
VHH4SfiFor     ____ SfiI____
CATGCCATGACTCGCGGCCCAGCCGGCC ATGGCCCAGGTGCAGCTGCAGGAGTCGGG
  M  P  -  L  A  A  Q  P  A   M  A  Q  V  Q  L  Q  E  S  

pPaniBsrG1
         BsrG1
AGCTGTCA TGTACA CTCCCAAGTTCAACTGCAAGAGTCTGGCCCTG
 S  C  H   V  H   S  Q  V  Q  L  Q  E  S  G  P  

p19BsrG1
AGCTGTCA TGTACA  CTCCCAAGTTCAACTTCAGCAGTCTGGCGC
 S  C  H   V  H   S  Q  V  Q  L  Q  Q  S  G   
pHer2BsrG1
AGCTGTCA TGTACAC TCCGAAGTTCAACTTGTGGAATCTGGCGGC
 S  C  H   V  H   S  E  V  Q  L  V  E  S  G  G  

p19BsrG1
         BsrG1             
AGCTGTCA TGTACAC TCCCAAGTTCAA CTTCAGCAGTCTGGCGC
 S  C  H   V  H   S  Q  V  Q   L  Q  Q  S  G  

pPaniBsrG1
AGCTGTCA TGTACA CTCCCAAGTTCAACTGCAAGAGTCTGGCCCT
 S  C  H   V  H   S  Q  V  Q  L  Q  E  S  G  P  
pHer2BsrG1
AGCTGTCA TGTACAC TCCGAAGTTCAACTTGTGGAATCTGGCGGC
 S  C  H   V  H   S  E  V  Q  L  V  E  S  G  G  

HCkRev 
AGACTCTCCCCTGTTGAAGCTCTT

sequencing primers
pADLFor
TTTACACTTTATGCTTCCGGCTCGTATGTTG
pADLRev
AGGTTTTGCTAAACAACTTTCAAC

pG2CdBssHII
GCCAGGCTCAAGGCCCGCATGCCCGACGGCGA

HuIgMRev
TGGAAGAGGCACGTTCTTTTCTTT
C_Rev
AGACTCTCCCCTGTTGAAGCTAAG
C_Rev
TGAAGATTCTGTAGGGGCCACTGTCTT

HuJH12Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCAGGGTGCC
ggcaccctggtcaccgtctcctcaggtggttcctctagatcttcc
 G  T  L  V  T  V  S  S  G  G  S  S  R  S  S  

HuJH3Rev
GGAAGATCTAGAGGAACCACCTGAAGAGACGGTGACCATTGTCCC
gggacaatggtcaccgtctcttcaggtggttcctctagatcttcc
 G  T  M  V  T  V  S  S  G  G  S  S  R  S  S  

HuJH45Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCAGGGTTCC
ggaaccctggtcaccgtctcctcaggtggttcctctagatcttcc
 G  T  L  V  T  V  S  S  G  G  S  S  R  S  S  

HuJH6Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCGTGGTCCC
gggaccacggtcaccgtctcctcaggtggttcctctagatcttcc
 G  T  T  V  T  V  S  S  G  G  S  S  R  S  S  

pCHIBack
GCTCGGGGAAGTAGTCCTTCACCAGG

pCKBACK
TTCCAGATTTCAACTGCTCATCAG

HuVH1B7AFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTCAGRTGCAGCTGGTGCARTCTGG
tactcgcggcccagccggccatggctcagrtgcagctggtgcartctgg
   L  A  A  Q  P  A  M  A  Q  X  Q  L  V  X  S   
    
recombinant single chain antibody pIII fusion https://www.ncbi.nlm.nih.gov/protein/CAA74922
MKYLLPTAAAGLLLLAAQPAMAQ
VQLQQSGAELVRPGVSVKISCKGSGYKFTDYATHWVKQSHAKSLEWIGVISTYYGDTTYNQKFKGKATMTVDKSSSTAYMELPRLTSDDSAIYYCALLRPFAYWGQGTTVTVSSGSASAPKLEEGEFSEARVDIVLTQSPLSLSVSAGEKVTMSCKSSQSLLNSGNQNNDLAWYQQKPGQRPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIKRAAAGSKDIRAETVESCLAKSHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENE

GGGSEGGGSEGGGSEGGG

TKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDLFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES

https://www.ncbi.nlm.nih.gov/nuccore/20797177
https://www.ncbi.nlm.nih.gov/protein/CAD91925 anti-human CD28 
  sig_peptide 
  1 metdtlllwv lllwvpgstg 

IgV_L_kappa
  divltqspas lavslgqrat iscrasesve yyvtslmqwy
       61 qqkpgqppkl lifaasnves gvparfsgsg sgtnfslnih pvdeddvamy fcqqsrkvpy
      121 tfgggtklei kr

      ggggsggg gsggggsqvk 
IgV_H
      lqqsgpglvt psqslsitct vsgfslsdyg
      181 vhwvrqspgq glewlgviwa gggtnynsal msrksiskdn sksqvflkmn slqaddtavy
      241 ycardkgysy yysmdywgqg ttvtvs

      sast kgpsvfplap sssgsgqvk
IgV_H
      lqqsgaelvrp
      301 gasvklscka sgytftnywi nwvkqrpgqg lewigniyps yiytnynqef kdkvtltvde
      361 ssstaymqls sptsedsavy yctrspygyd eygldywgqg ttvtv

      ssggg gsggggsggggs
IgV_L_kappa
      dieltqsp ssltvtagek vtmnckssqs llnsrnqkny ltwyqqkpgq ppklliywas
      481 tresgvpdrf tgsgsgtdft ltissvqaed lavyycqndy vypltfgagt klei

      krtvaa
      541 psvfifppsa eeqkliseed lnsavd

HuVH1CFor
       ____ SfiI____ 
TACTCGCGGCCCAGCCGGCCATGGCTSAGGTCCAGCTGGTRCAGTCTGG
                     M  A  X  V  Q  L  X  Q  S   
HuVH2BFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTCAGRTCACCTTGAAGGAGTCTGG
                     M  A  Q  X  T  L  K  E  S   
HuVH3BFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTSAGGTGCAGCTGGTGGAGTCTGG
                     M  A  X  V  Q  L  V  E  S   
HuVH3CFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTGAGGTGCAGCTGGTGGAGWCYGG
                     M  A  E  V  Q  L  V  E  X   
HuVH4BFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTCAGGTGCAGCTACAGCAGTGGGG
                     M  A  Q  V  Q  L  Q  Q  W   
HuVH4CFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTCAGSTGCAGCTGCAGGAGTCSGG
                      M  A  Q  X  Q  L  Q  E  X   
HuVH5BFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTGARGTGCAGCTGGTGCAGTCTGG
                     M  A  X  V  Q  L  V  Q  S   
HuVH6AFor
       ____ SfiI____
TACTCGCGGCCCAGCCGGCCATGGCTCAGGTACAGCTGCAGCAGTCAGG
                     M  A  Q  V  Q  L  Q  Q  S    

Light chain Forward primers
HuV_5For
GGTGGCGGTGGCTCGGGCGGTGGTGGG GAAACGACACTCACGCAGTCTCC
 G  G  G  G  S  G  G  G  G   E  T  T  L  T  Q  S    
HuV_6For
GGTGGCGGTGGCTCGGGCGGTGGTGGG GAAATTGTGCTGACTCAGTCTCC
 G  G  G  G  S  G  G  G  G   E  I  V  L  T  Q  S  

HuV_1AFor                   light chain lambda 
GGTGGCGGTGGCTCGGGCGGTGGTGGG CAGTCTGTGCTGACTCAGCCACC
 G  G  G  G  S  G  G  G  G   Q  S  V  L  T  Q  P   

HuV_1BFor
GGTGGCGGTGGCTCGGGCGGTGGTGGG CAGTCTGTGYTGACGCAGCCGCC
 G  G  G  G  S  G  G  G  G   Q  S  V  X  T  Q  P    

HuV_2For
GGTGGCGGTGGCTCGGGCGGTGGTGGG CARTCTGCCCTGACTCAGCCT
 G  G  G  G  S  G  G  G  G   X  S  A  L  T  Q  P  
HuV_3AFor
GGTGGCGGTGGCTCGGGCGGTGGTGGG TCCTATGWGCTGACTCAGCCACC
 G  G  G  G  S  G  G  G  G   S  Y  X  L  T  Q  P    

HuV_3BFor
GGTGGCGGTGGCTCGGGCGGTGGTGGG TCTTCTGAGCTGACTCAGGACCC
 G  G  G  G  S  G  G  G  G   S  S  E  L  T  Q  D    

HuV_4For
GGTGGCGGTGGCTCGGGCGGTGGTGGG CACGTTATACTGACTCAACCGCC
 G  G  G  G  S  G  G  G  G   H  V  I  L  T  Q  P   

HuV_5For
GGTGGCGGTGGCTCGGGCGGTGGTGGG CAGGCTGTGCTGACTCAGCCGTC
 G  G  G  G  S  G  G  G  G   Q  A  V  L  T  Q  P    

HuV_6For
GGTGGCGGTGGCTCGGGCGGTGGTGGG AATTTTATGCTGACTCAGCCCCA
 G  G  G  G  S  G  G  G  G   N  F  M  L  T  Q  P    

HuV_78For
GGTGGCGGTGGCTCGGGCGGTGGTGGG CAGRCTGTGGTGACYCAGGAGCC
 G  G  G  G  S  G  G  G  G   Q  X  V  V  X  Q  E    

HuV_9For
GGTGGCGGTGGCTCGGGCGGTGGTGGG CWGCCTGTGCTGACTCAGCCMCC
 G  G  G  G  S  G  G  G  G   X  P  V  L  T  Q  X    

LinkerFor, verified         anneal to Light chain Forward primers
GGTGGTTCCTCTAGATCTTCCTCCTCT GGTGGCGGTGGCTCGGGCGGTGGTGGG
 G  G  S  S  R  S  S  S  S   G  G  G  G  S  G  G  G  G  

HuJ_1RevSfiI, verified
       ____ SfiI____
TGGTGTTGGCCTCCCGGGCCACTAGTACGTTTGATHTCCASYTTGGTCCC
                                ____ SfiI____
(GGGACCAARSTGGADATCAAACGTACTAGT GGCCCGGGAGGCCAACACCA)
  G  T  X  X  X  I  K  R  T  S   G  P  G  G  Q  H                                 


HuJ_1_2RevSfiI, verified
       ____ SfiI____
TGGTGTTGGCCTCCCGGGCCACTAGTACCTAGGACGGTGACCTTGGTCCC
                                ____ SfiI____
(GGGACCAAGGTCACCGTCCTAGGTACTAGT GGCCCGGGAGGCCAACACCA)
  G  T  K  V  T  V  L  G  T  S   G  P  G  G  Q  H    

HuJ_2RevSfiI, verified
       ____ SfiI____
TGGTGTTGGCCTCCCGGGCCACTAGTACGTTTAATCTCCAGTCGTGTCCC
                                ____ SfiI____
(GGGACACGACTGGAGATTAAACGTACTAGT GGCCCGGGAGGCCAACACCA)
  G  T  R  L  E  I  K  R  T  S   G  P  G  G  Q  H

HuJ_2_2RevSfiI, verified
       ____ SfiI____
TGGTGTTGGCCTCCCGGGCCACTAGTACCTAGGACGGTCAGCTTGGTCCC
                                ____ SfiI____
(GGGACCAAGCTGACCGTCCTAGGTACTAGT GGCCCGGGAGGCCAACACCA)
  G  T  K  L  T  V  L  G  T  S   G  P  G  G  Q  H

HuJ_3RevSfiI, verified
       ____ SfiI____
TGGTGTTGGCCTCCCGGGCCACTAGTACYTAAAACGGTGAGCTGGGTCCC
                                ____ SfiI____
(GGGACCCAGCTCACCGTTTTARGTACTAGT GGCCCGGGAGGCCAACACCA)
 G  T  Q  L  T  V  L  X  T  S    G  P  G  G  Q  H    


====
Human VH BackSfiI primers
VH1b/7aBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAG(AG)TGCAGCTGGTGCA(AG)GG-3'
VHLCBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC(GC)AGGTCCAGCTGGT(AG)CAGTCTGG-3'
VH2bBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAG(AG)TCACCTTGAAGGAGTCTGG-3'
VH3bBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC(GC)AGGTGCAGCTGGTGGAGTCTGG-3'
VH3cbackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAG(AT)C(TC)GG-3'
VH4bBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTACAGCAGTGGGG-3'
VH4cBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAG(GC)TGCAGCTGCAGGAGTC(GC)GG-3'
VH5bBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGA(AG)GTGCAGCTGGTGCAGTCTGG-3'
VH6aBackSfiI
5'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGG-3'

Human JHforXhoI primers
HuJHFor1-2XhoI
5'-ACCGCCTCCACCACTCGAGACGGTGACCAGGGTGCC(TC)(TC)(GT)GCCCCA-3'
HuJHFor3XhoI
5'-ACCGCCTCCACCACTCGAGACGGTGACCATTGTCCC(TC)(TC)(GT)GCCCCA-3'
HuJHFor4-5XhoI
5'-ACCGCCTCCACCACTCGAGACGGTGACCAGGGTTCC(TC)(TC)(TG)GCCCCA-3'
HuJHFor6XhoI
5'-ACCGCCTCCACCACTCGAGACGGTGACCGTGGTCCC(TC)(TC)(TG)CCCCCA-3'

HumanVBackApaLIPrimers
Huλ1aBackApaLI
5'-ACCGCCTCCACCAGTGCACAGTCTGTGCTGACTCAGCCACC-3'
Huλ1bBackApaLI
5'-ACCGCCTCCACCAGTGCACAGTCTGTG(TC)TGACGCAGCCGCC-3'
Huλ1cBackApaLI
5'-ACCGCCTCCACCAGTGCACAGTCTGTCGTGACGCAGCCGCC-3'
Huλ2BackApaLI
5'-ACCGCCTCCACCAGTGCACA(AG)TCTGCCCTGACTCAGCCT-3'
Huλ3aBackApaLI
5'-ACCGCCTCCACCAGTGCACTTTCCTATG(AT)GCTGACTCAG
CCACC-3'
Huλ3bBackApaLI
5'-ACCGCCTCCACCAGTGCACTTTCTTCTGAGCTGACTCAGGACCC-3'
Huλ4BackApaLI
5'-ACCGCCTCCACCAGTGCACACGTTATACTGACTCAACCGCC-3'
Huλ5BackApaLI
5'-ACCGCCTCCACCAGTGCACAGGCTGTGCTGACTCAGCCGTC-3'
Huλ6BackApaLI
5'-ACCGCCTCCACCAGTGCACTTAATTTTATGCTGACTCAGCCCCA-3'
Huλ7/8BackApaLI
5'-ACCGCCTCCACCAGTGCACAG(AG)CTGTGGTGAC(TC)CAGGAGCC-3'
Huλ9BackApaLI
5'-ACCGCCTCCACCAGTGCAC(AT)GCCTGTGCTGACTCAGCC(AC)CC-3'

HumanVκBackApaLIPrimers
Huκ1bBackApaLI
5'-ACCGCCTCCACCAGTGCACTTGACATCCAG(AT)TGACCCAG
TCTCC-3'
Huκ2BackApaLI
5'-ACCGCCTCCACCAGTGCACTTGATGTTGTGATGACTCAGTCT
CC-3'
Huκ3bBackApaLI
5'-ACCGCCTCCACCAGTGCACTTGAAATTGTG(AT)TGAC(AG)CAG
TCTCC-3'
Huκ4b BackApaLI
5'-ACCGCCTCCACCAGTGCACTTGATATTGTGATGACCCACACT
CC-3'
Huκ5 BackApaLI
5'-ACCGCCTCCACCAGTGCACTTGAAACGACACTCACGCAGTCT
CC-3'
Huκ6 BackApaLI
5'-ACCGCCTCCACCAGTGCACTTGAAATTGTGCTGACTCAGTCT
CC-3'
Human J􀁨 ForNotI Primers
HuJ􀁨1 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTT
GGTCCC-3'
HuJ􀁨2-3 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTT
GGTCCC-3'
HuJ􀁨4-5 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACTTAAAACGGTGAGCTG
GGTCCC-3'
Human Jκ ForNotI Primers
HuJκ ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTT
GGTCCC-3'
HuJκ2 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTT
GGTCCC-3'
HuJκ3 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTT
GGTCCC-3'
HuJκ4 ForNotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCACCTT
GGTCCC-3'
HuJκ5 FornotI
5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCG
TGTCCC-3'

Primers for Recovery of VH and Linker Sequence
pUC19 Rev Primer
5'-AGCGGATAACAATTTCACACAGG-3'
fdtetseq Primer
5'-GTCGTCTTTCCAGACGTTAGT-3'

Oligonucleotide Primers for Pull-Through PCR

JH for Primers
HuJH1-2For
5'-TGAGGAGACGGTGACCAGGGTGCC-3'
HuJH3For
5'-TGAAGAGACGGTGACCATTGTCCC-3'
HuJH4-5For
5'-TGAGGAGACGGTGACCAGGGTTCC-3'
HuJH6For
5'-TGAGGAGACGGTGACCGTGGTCCC-3'
Rev JH Primers
RHuJH1-2
5'-GCACCCTGGTCACCGTCTCCTCAGGTGG-3'
RHuJH3
5'-GGACAATGGTCACCGTCTCTTCAGGTGG-3'
RHuJH4-5
5'-GAACCCTGGTCACCGTCTCCTCAGGTGG-3'
RHuJH6
5'-GGACCACGGTCACCGTCTCCTCAGGTGC-3'

</pre>
[[Category:Antibodies]]