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ScFv Library Construction Protocol

Antibody scFv fragments (single-chain Fv) are popular recombinant antibody formats and the smallest antibody fragments capable of recognizing an antigen, but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and is also applicable for stability selection. ScFv is well displayed on filamentous phage and is often the preferred antibody format in antibody engineering. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or chaotropic agents. ScFv molecules combine the coding sequence of the variable heavy (VH) and sequence of the variable light chain (VL) domains of an antibody in a single-gene encoded format. The resulting polypeptides, with the variable light (VH ) and heavy chain (VH ) domains connected by a flexible peptide linker, were assemble into functional antigen-binding sites.

The orientation of ligation of VL and VH is VL-linker- VH  or VH-linker- VL. The antigen specificity of the two construction types is similar but they can lead to secretory expression in E.coli in different level. So you can choose any of the two construction orientations and then design primer by PCR method and sequence assembly type. Several PCR strategies are listed below:

  1. Normal PCR strategy: Design primer (introduce restriction enzyme sites) for amplifying VH and VL gene based on antibody CDR sequence. Link C-terminal of VH and N- terminal of VL, or C-terminal of VL and N- terminal of VH by linker (design primer also based on sequence of VH, VL and linker. Choose appropriate restriction enzyme to gain the goal of sequence assembly. You can choose any assembly fragment types to clone, eg,. a. Design primers for amplifying VH gene and linker-VL. b. Design primers for amplifying VH-linker gene and VL) to construct antibody fragment or gene fragment. The suitable that the length of linker is 15-20 amino acids.
  2. Pull-through PCR strategy: To avoid non-specific binding or reducing PCR efficiency. You can choose this method to introduce restriction sites on the end of a PCR product. The pull-through method performs a PCR like normal and you purify the PCR product from a gel. From this PCR product, you then run a few additional PCR steps with primers that have your restriction sites on the ends. If there is restriction enzyme site in your target VH or VL gene, it is suggested to use this method for getting ligation PCR product and digested vector.
  3. Slicing by overlap extension PCR strategy: Don’t need to introduce restriction enzyme site into primers for ligation of VH, linker and VL. ScFv fragments were assembled by this method. After assembly, the scFv fragments (VL- linker - VH or VH - linker - VL ) were further amplified and, concurrently, longer tails were added to the ends in order to facilitate the subsequent digestion of the fragments by restriction enzymes. Then digest phagemid vectors by the same enzyme for combining them with scFv fragment .
  4. RACE (rapid amplication cDNA) PCR strategy: This technique can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) of the RNA. Add universal linker primers in mRNA transcription, gain unknown sequence of cDNA by gene specific primer.

Here, we describe a basic protocol concluded by Simon Lennard for the construction of scFv gene libraries (naïve libraries) by pull-through PCR strategy and phage display. The basic protocol flow chart is shown in Fig.1.

Protocol flow chart Fig.1 Protocol flow chart.

Materials

  1. Fresh source of lymphoid tissue from which mRNA can be isolated.
  2. Kit for the preparation of mRNA (e.g., an oligo (dT)-purification system).
  3. Kit for the synthesis of cDNA (e.g.,“First-strand cDNA synthesis kit”).
  4. PCR reagents: Taq DNA polymerase with 10x Taq DNA polymerase buffer, a stock of deoxyribonucleoside triphosphates (dNTPs). PCR H2O [ACS] reagent;
  5. Oligonucleotide primers (design based on your requirement) at 10 μM.
  6. Kit for the isolation of DNA from gels.
  7. Phage display vector(s) and appropriate restriction enzymes for cloning.(Here we represent for vector 1 and vector 2,enzyme A, B, C, D,E)
  8. Kit for medium-scale isolation of plasmid DNA.
  9. Ligation kit, 5x ligation buffer (500 mM Tris-HCl, 25 mM MgCl2, pH7.4).
  10. Electrocompetent Escherichia coli strain(e.g., TG1).
  11. Gene Pulser and suitable electroporation cuvets.
  12. 2TY as liquid and solid media.
  13. Glucose 20% (w/v) sterile-filtered.
  14. Filtersterilized antibiotic for screening positive clones (e.g., Ampicillin (100 mg/mL stock) and kanamycin (50 mg/mL stock)) .
  15. Kit for the purification of PCR products.
  16. 20% (w/v) Polyethylene glycol (PEG) 8000, 2.5 M NaCl.
  17. TE buffer (10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid, pH 8.0).
  18. Cesium chloride (CsCl).

Methods

First strand cDNA synthesis from PBLC mRNA

PBLC (peripheral blood lymphocytes cell) isolation and synthesis of primary cDNA template. Total RNA was prepared from PBMC by using a RNA Purification kit. First strand cDNA was synthesized from total RNA by using a First-Strand cDNA Synthesis kit. Follow the manufacturer’s instructions.
(Note: Rapid processing of fresh tissue samples is essential if the full diversity of the Ab repertoire is to be recovered. Tissue, mRNA, or cDNA product can be stored at -70°C, if some loss of diversity is acceptable (perhaps libraries from infected or immunized individuals, rather than a comprehensive naive library). 50 mL of blood should yield approx 1 x 107 cells, which in turn yield about 10 μg total RNA, of which 1–5% is mRNA. It is important to ensure that there is enough cDNA for all the VH and VL PCR reactions planned, each of which requires 0.5 ng cDNA.)

Amplification of Antibody Variable Region Genes: The goal of this process is gain VH and linker-VL gene sequence.

VH Repertoire Construction

  1. VH gene repertoires were PCR amplified by using the cDNA prepared from above procedure. To amplify the VH genes from the cDNA and plasmid template, the primers were designed based on what described on pull-through PCR strategy. Perform PCR reactions for each VH primers using the optimizing cycling parameters by PCR reagents.
  2. Digest the purified VH DNA and vector 1 with two required enzymes (enzyme A and enzyme B) respectively using the reaction buffer provided by the providers. After digestion, heat-inactivate the enzyme at proper temperature.
  3. Purify digesting product of VH DNA and vector 1 respectively by Kit for the purification of PCR products..
  4. Ligate purified product of VH DNA and vector 1 by Ligation kit.
    (Note: 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 vector 1 within the range specified by the manufacturer of the ligation kit. A “vector only” ligation control should be included to determine the background caused by nonrecombinants.)
  5. Transformation: Electroporate the DNA ligations into competent E. coli. 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.
  6. Plate aliquots from each transformation to 2TYG plates containing antibiotic (e.g., ampicillin 100 μg/mL; 2TYAG) and incubate overnight at 30°C.
    (Note: Confirm that background levels of vector self-ligation are minimal and which ligation ratio yields the highest numbers of transformants.)
  7. From the planned size of the VH library 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 4). Go on performing electroporations recovery and determine the library size by serial dilutions.
  8. Perform at least 40 electroporations with electrocompetent E. coli cells (step 5), 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. Scrape cells from the large plates and prepare glycerol stocks for storage at -70°C.

VL Repertoire Construction (Note: VL kappa and VL lamda gene fragments are amplified separately using each back primer in combination with the appropriate equimolar mixture of the J kappa or J lamda forward primers. After recovery of the combined VL repertoire, the next stage is to clone in the (Gly4Ser)3 scFv linker from an existing scFv, together with a dummy VH, recovered by PCR from an irrelevant clone.)

  1. Perform PCR reactions using the same cycling parameters as outlined for recovery of VH products (see VH repertoire construction step 1).
  2. Digest the purified VL DNA and vector 2 respectively by two required enzymes (C and D restriction enzymes).
  3. Perform ligation reactions and subsequent electroporations as described for the VH repertoire (see VH repertoire construction). The size of the VH and VL repertoires can be estimated.
  4. To introduce the scFv-linker (e.g., (Gly4Ser)3) into the finished VL repertoire, PCR amplify the linker from an existing scFv, together with an irrelevant (dummy) VH fragment. Digest linker single product with enzyme E. Get Purified product described previously (see VH repertoire construction). And then estimate its concentration by comparison with DNA markers.
  5. Prepare the vector 2 containing the VL repertoire on mini prep scale and sequentially digest with enzyme D and enzyme E.
  6. Perform sufficient ligation reactions to dummy VH-linker DNA fragment into a pool of the VH and VL libraries.
  7. Transformation: Electroporate into E. coli cells, and plate out as described previously (see VH repertoire construction).

Construction of The ScFv Library: The amplified VH and linker-VL genes were gel-purified on agarose, the scFv assembly and expression vector were digested and ligated to gain scFv library.

  1. Amplify the VH and linker-VL DNA fragments separately from each of the cloned repertoires. Perform PCR reactions using the cycling parameters described previously (see VH repertoire construction).
  2. To perform the assembly reaction, add the required reagents to the pooled VH and linker-VL products, and perform optimizing cycling parameters.
  3. Prepare pull-through PCR reactions. Replicates of each reaction are advisable, to maximize the diversity of the final library. Using assembly DNA reaction, amplify with cycling parameters described previously (see VH repertoire construction). The correct size of the assembled construct is must be known.
  4. Digest purified PCR product with B and D restriction endonucleases (need to design linker-VL amplifying primers) as described previously (see VH repertoire construction and VL repertoire construction).
  5. Gel-purify the digested scFv assembly construct and ligate with B/D digested vector 1 after determining the optimum insert vector ratio as described previously (see VH repertoire construction). Perform at least 100 electroporations, pool into batches, and plate out each batch on 2TYAG (2TYG containing antibiotics) plates. Determine the total size of the library by taking aliquots from each batch and plating out serial dilutions on 2TYAG (2TYG containing antibiotics).
  6. Scrape cells from the large plates and prepare glycerol stocks for storage at -70°C.

Preparation of Library Phage

  1. Inoculate some 2TYG with certain number of cells from the library glycerol stock and incubate at 37°C in a shaker.
  2. Add at least 10x excess of M13KO7 helper phage to a final concentration of 5 x 109 pfu/mL, and incubate in shaker to allow phage infection.
  3. Resuspend the pellet in the same volume of 2TYAG (2TYG containing antibotics). Incubate overnight with rapid shaking.
  4. Pellet the cells and recover the supernatant containing the phage into prechilled bottles. Add 1/3 volumes of PEG/NaCl to the supernatant Mix gently and allow the phage to precipitate for 1 h on ice.
  5. Pellet the phage by twice centrifuging in the same bottle at 4°C. Carefully remove all the supernatant and resuspend the phage pellet TE buffer.
  6. Recentrifuge the phage in smaller tubes and recover the supernatant, which will now contain the phage. Remove any particulate material and ensure that any bacterial pellet that appears is left undisturbed.
  7. Add caesium chloride to the phage suspension. Using an ultracentrifuge, spin the samples.
  8. Dialyze the phage against two changes of 1 L TE.
    (Notes: The resultant phage are purified by PEG precipitation and caesium-banding, and, as a result, are stable at 4°C for 2 year. Phage prepared by PEG precipitation alone should only be stored at 4°C for 1–2 week)
  9. Finally, titer phage stocks by infecting E. coli cells with dilutions of phage stock, plating to 2TYA, incubation, and enumeration of the numbers of antibiotic resistant colonies that appear. The phage can then be stored in aliquots at 4°C for long periods, ready for screening.

Here, we only provide a basic procedure of scfv library construction by pull-through PCR for your reference. However, it is a complicated process that researchers are spending so much time to probe the best condition for each individual project.

If you need help or have any questions about the protocol described above, please feel free to email us at info@creative-biolabs.com or call us on 1-631-871-5806 anytime.

Reference:
  1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1990) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

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