Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly
3.2 Materials
[edit]- 5 x 106 cells from a growing or frozen hybridoma culture or spleen cells,respectively
* PCR primers (Figs. 3.2 and 3.3) and corresponding plasmids (Figs. 3.4 and 3.5)
* Helper phage (e.g., Stratagene VCSM13 # 200251)
* F+, supE, recA strain (e.g., E. coli XL1-Blue) (available in electrocompetent/chemocompetent form from Stratagene)
* Anti-M13 antibody HRP-conjugate (GE Healthcare; # 27-9421-01)
* PEG 6000 (Fluka)
* Sterile, RNase-free equipment: pipet tips, tubes, RNase-free ultra high purity (UHP) water, baked nondisposable glassware, and sterile, disposable plasticware
* Standard molecular biology equipment and reagents for:
– Determining the isotype of mAbs (Roche IsoStrip Mouse Monoclonal Antibody Isotyping Kit)
– Purifying RNA (Invitrogen TRIzol reagent and Qiagen RNeasy Mini Kit)
– Performing a cDNA synthesis reaction (Qiagen QuantiTect Reverse Transcription Kit)
– Performing PCR reactions
– Purifying PCR products (Macherey Nagel PCR clean-up Gel Extraction Kit)
– Cutting and gel-purifying DNA (Sigma-Aldrich GenElute Gel Extraction Kit)
– Concentrating DNA (Amicon Microcon 30 for volumes less than 500 ml)
– Ligating and transforming DNA
– Growing bacteria and phages
– Conducting an Enzyme Linked Immunosorbent Assay (ELISA)
– Performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting.
3.3 Method
[edit]3.3.1 Isolation of mRNA and cDNA Synthesis
[edit]1. Take 5 x 106 cells from a frozen or growing hybridoma culture (for isotype determination, use the Roche IsoStrip Mouse Monoclonal Antibody Isotyping Kit) or spleen cells, respectively (see note). Perform a total RNA preparation, combining homogenization of cells in the presence of TRIzol Reagent (Invitrogen) with RNA purification using the Qiagen RNeasy Mini Kit as described by the manufacturers. According to the supplier, the latter kit can be used for up to 1 x 107 cells, but in order to get highly pure mRNA, take only 5 x 106 cells.
Note: For RNA preparation from mouse spleen (typically yielding 5 x 107 B-cells each), first separate it from connective tissue with sharp forceps or scissors (if frozen, also cut the frozen tissue into smaller pieces and pulverize using a mortar) and homogenize it using the Tissue Lyser (Qiagen) or similar homogenizers in the presence of 1 ml TRIzol Reagent per 50 mg of tissue. Make sure not to use too many cells as spleens are typically rich in nucleases, and, therefore, enough RNAse-deactivating components from the TRIzol Reagent should be present in the solution. TRIzol Reagent is a commercial monophasic preparation of guanidinium isothiocyanate and phenol and only the addition of chloroform separates the solution in two phases. If desired, polyA+ mRNA can subsequently be isolated from the total RNA using the Oligotex Direct mRNA Mini Kit (Qiagen) – however, in most cases, this should not be necessary for the
subsequent production of cDNA. Therefore, we do not recommend including this additional purification step, as it might lead to loss of mRNAs present only in smaller quantities. Since specific amplification primers are used, we consider it rather advantageous to work with a higher total amount of RNA.
2. Separate the RNA from DNA and proteins by phenol-chloroform extraction with subsequent silica membrane purification as described by the manufacturer (Invitrogen). Transfer the upper aqueous phase to a new, RNase-free tube. Add an equal volume of 100% ethanol dropwise, as its presence is required for the RNeasy columns to bind the RNA during the initial application. Transfer up to 700 µl of the mixture, including any precipitate that may have formed, to one RNeasy spin column and continue according to the manufacturer’s instructions.
Note: RNA in harvested tissue is not protected from degradation until the sample is mixed with TRIzol Reagent, flash-frozen or disrupted and homogenized in the presence of RNase-inhibiting or protein-denaturing reagents. Therefore, proceed with this step as fast as possible. Generally, DNase digestion is not required with RNeasy Kits since its silica membrane efficiently removes most of the DNA. However, if desired, residual DNA can be removed by optional oncolumn DNase digestion using the RNase-Free DNase Set (Qiagen). It is
important not to overload the RNeasy spin column, as this will significantly reduce RNA yield and quality.
3. Elute the purified RNA by the addition of 30 µl RNase-free water. The mRNA solution is now ready for cDNA synthesis or can alternatively be stored at -80°C for up to one month.
Note: Diethylpyrocarbonate (DEPC)-treated UHP water can also be used. However, as DEPC is a suspected carcinogen the use of filtrated RNase-free water is recommended.
4. For reverse transcription, take approximately 0.1–0.5 µg RNA and 1 µl random hexamer primers provided in the kit in 20 µl total reaction volume. The precise
procedure is described in the QuantiTect Reverse Transcription Kit (Qiagen).
Note: Ribonuclease H activity of Quantiscript Reverse Transcriptase specifically degrades only the RNA in RNA:DNA hybrids, but it has no effect on pure RNA. Hence, an additional RNA degradation step using another RNase H enzyme is not necessary prior to subsequent PCR reaction.
Note: Specific primers hybridizing to the constant regions can be used as well, e.g., if only a particular antibody class should be amplified from spleen cells. In general, however, the random hexamer primers work robustly.
3.3.2 PCR Amplification and scFv Assembly
[edit]
3.3.2.1 PCR Amplification of VL and VH Domains
[edit]
1. Use the primers described in Fig. 3.2, which have been dissolved in 100 µM stock solutions in either sterile water or sterile TE buffer to prepare appropriate
mixtures (VL-for mix, VL-rev mix, VH-for mix, and VH-rev mix). Mix them according to the degree of degeneration, indicated as “d” in Fig. 3.2 (equaling the number of different unique sequences encoded by mixed bases in the primer) by adding the stated volumes (in ml) towards the final primer mix. The fraction of lambda-specific primers in both the forward and reverse VL mixture amounts for ~5% of the total volume, accounting for the low percentage of this light chain type in mouse antibodies. The nominal total primer concentration of these
mixtures is still 100 µM, ranging from 3 to 40 µM for each of the individual oligonucleotides.
Note: As described in the introduction, problems in the cloning of monoclonal antibodies can occur if the hybridoma transcribes more than one functional or even nonfunctional heavy or light chain variable region gene. Therefore, it is highly recommended to omit any lambda chain-specific primer in the PCR if the isotyping already indicates that the hybridoma of interest secretes IgGs possessing kappa light chains.
2. For PCR amplification of VL and VH, use the product of the completed firststrand cDNA reaction and prepare the following mixtures:
| Components | PCR mix for amplification of VL |
PCR mix for amplification of VH |
| cDNA |
2 µl |
2 µl |
| dNTPs (10 mM each) |
1 µl |
1 µl |
| 5 x Phusion buffer (NEB) |
10 µl |
10 µl |
| primer mix (100 mM) |
1 µl VL (or 0.5 µl ) |
1 µl VH (or 0.5 µl ) |
| primer mix (100 mM) |
1 µl VL-rev (or 0.5 µl ) |
1 µl VH-rev (or 0.5 µl ) |
| DMSO |
2.5 µl |
2.5 µl |
| Phusion Polymerase, 2 U/µl | 1 µl |
1 µl |
| H2O |
30.5 µl |
30.5 µl |
| Total | 50 µl | 50 µl |
Note: This standard protocol is optimized for VentR polymerase, a DNA polymerase with a 5–15 fold lower error rate than Taq DNA Polymerase (due to an intrinsic 30!50 proofreading exonuclease activity). If using other proofreading polymerases (e.g., Phusion High-Fidelity DNA Polymerase from Finnzymes), reaction and PCR program conditions might have to be adapted. If the proposed PCR mix does not lead to any product, varying the cDNA template amount might be beneficial. If the thermocycler does not have a heated cover, add one
drop of mineral oil to the reaction tube to prevent evaporation.
3. Performthe following PCRcycles after an initial denaturation of the DNA template for 3 min at 95°C: 5 cycles of 30 s at 95°C, 30 s initial annealing at55°C, and 45 s elongation at 72°C, followed by 20 cycles of 30 s at 95°C, 30 s at 63°C (or 55 °C), and 45 s at 72°C. After the last cycle is completed, an additional 5 min elongation step at 72°C should be performed before cooling the thermocycler to 4°C.
Note: We recommend using a hot start, keeping the PCR tubes on ice and not placing them into the thermocycler until the block has reached 95°C, to minimize
unspecific amplification. For successful amplification of VL and VH, complete annealing of the 30-ends of the primers with the template DNA is essential. The recommended annealing temperature of 55°C should be suitable for approx. 97% of the sequences found in a reference set of murine germline sequences in the IMGT database. However, as it is not clear a priori which somatic mutations a given monoclonal antibody may carry in the primer regions, we recommend using a gradient PCR program (covering a range between 70°
and 50°C in steps of 2°) to determine the optimum annealing temperature and to amplify the antibody genes without unspecific secondary bands. Alternatively, the PCR might also be run in a “touchdown” manner (Don et al. 1991), starting at an annealing temperature of70°C and ending at 50°C. As after 5 cycles the amplified PCR product will serve itself as template DNA, the annealing temperature of the last 20 cycles can be increased to 63°C.
4. Analyze 1/10 volume of each PCR mixtures by agarose gel electrophoresis, purify the VL and VH genes using the PCR clean-up Gel Extraction Kit (Macherey Nagel) according to the manufacturer’s instructions and determine the DNA concentration of both genes.
Note: Using the listed primer mixtures, the expected lengths of the PCR products of VL and VH are between 375–402 bp and 386–440 bp, respectively.
Purification of the PCR products is important to remove any residual primers which might interfere with the subsequent assembly PCR. For the case of multiple bands on the agarose gel, gel-purify the band of correct size using the GenElute Gel Extraction Kit (Sigma-Aldrich). If the final DNA concentration is too low afterward, perform a second PCR using these purified fragments as template for gaining sufficient yields of high-quality DNA.
3.3.2.2 Assembly of VL and VH by SOE-PCR (Splicing by Overlap Extension)
[edit]
1. For the assembly PCR, use approximately 10 ng of the PCR product of both domains in a total volume of 50 µl, containing 200 µM dNTPs, 3–5% DMSO,1 µMouter-for, and outer-rev primer (each) and 1 unit Phusion DNA Polymerase (NEB). Following a 3 min 95°C step, perform 5 cycles of 1 min at 95°C, 1 min at 63°C, and 1 min at 72°C, followed by another 5 cycles of 1 min at 95°C, 30 s at 56°C, and 1 min at 72°C and finally 25 cycles of 1 min at 95°C, 90 s at 72°C.
Note: Hot start PCR and initial assembly of VL and VH in the absence of the primers is usually not necessary but can be performed. It is important to include DMSO in the PCR mix as well as to keep the primer concentration as low as indicated to prevent any risk of primer-dimer formation.
Note: The assembly, as used here, places VL in front of VH. This has the advantage of placing a shortened FLAG tag, consisting of only four amino acids, at the N-terminus of the construct. Since its last amino acid, Asp, is the same as the first residue of the VL domain, only three additional amino acids are needed (Knappik and Plu¨ckthun 1994) for allowing specific detection using this tag. A slight asymmetry in the VH/VL heterodimer with respect to the pseudo two-fold axis (Plu¨ckthun et al. 1996) is taken care of with a 20-amino acid linker, leading
to monomeric scFv fragments.
3.3.3 Digestion and Cloning of scFv Genes
[edit]
1. Purify the product of the assembly PCR using the PCR clean-up Gel Extraction Kit (Macherey Nagel) according to the manufacturer’s instructions, eluting the product in 30 µl of the recommended buffer. In case there are several bands on the analytical agarose gel, carry out a gel purification of the correct band, as described in 3.3.3.4.
2. Perform a SfiI digest of the amplified scFv gene for3–4 h at 50°C (At 37°C, the activity of SfiI would be 10 fold-lower). To the 30 µl purified PCR product, add
5 µl 10 x NEbuffer 4 (NEB), 5 µl 10 x BSA (final concentration, 100 µg/ml), 9 µl H2O, and 1 µl (=20 units) SfiI (NEB).
3. Digest appropriate amounts of vector (pADL-23c, pAK100 or pJB12, see Fig. 3.4) with SfiI in the presence of the above-mentioned buffer, including BSA. Use 10 units SfiI
for 1 µg vector in 50 µl and incubate 4 h at 50°C. Dephosphorylate the cut vector by adding Calf Intestinal Alkaline Phosphatase (CIP, NEB; 0.5 unit/mg vector) to the digestion mix after 2 h and continue incubation for another 2 h at 50°C.
Note: Dephosphorylation should not be necessary because of the asymmetric overhangs. However, we always include this step to eliminate any risk of religation of single-cut vector.
Note: pAK100 or pJB12 are phage display vectors (Fig. 3.4). When starting from hybridomas, one can also directly clone the VL and VH genes into an scFv expression vector with a stronger promoter, such as pAK400, which does not encode a fusion with gIII. However, depending on the number of additional V genes expressed in the hybridoma, a large number of clones may have to be screened from individual colonies.
4. Purify the digested scFv antibody genes and vector by preparative agarose gel electrophoresis in combination with the GenElute Gel Extraction Kit (Sigma-Aldrich).
Note: For obtaining pure preparations of a fully digested vector, it is very important not to overload the agarose gel. Furthermore, the gel electrophoresis has to be run long enough to separate small amounts of undigested vector from the digested vector band. For large-scale vector or insert preparation, electroelution might be an efficient and convenient alternative. If the concentration of eluted DNA is too low for further applications, Microcon 30 columns (Amicon) can be used for concentration.
5. Ligate 50 ng scFv gene fragment with the vector (molar ratio of vector to insert 1:5) with 5 units T4 DNA ligase (NEB) in the presence of 1 x T4 DNA ligase buffer in 10 µl volume. Incubate for 2 h at room temperature or overnight at 16°C.
Note: The ATP-concentration is very crucial for the successful ligation by T4 DNA ligase. Therefore, we recommend using T4 DNA ligase buffer aliquots, which have been properly stored at -20°C and not thawed repeatedly. To allow an easy subcloning of the scFv fragment into vectors for optimized soluble expression and other purposes, compatible vector sets are available (Figs. 3.4 and 3.5).
6. Transform 100 µl chemocompetent TG1 (or XL1-Blue cells (Stratagene) with 10 µl of the ligation mix by heat-shock for 45 s at 42°C, add 500 µl of 2 x YT medium after 2 min incubation on ice, and incubate for 45 min, shaking at 37°C.
Note: Make sure not to exceed a ratio of ligation mix/cells of 1:10 (v/v). Chemocompetent E. coli are used, if only a very small diversity of clones is expected, e.g., when cloning from a hybridoma. If a larger diversity and thus many clones are required (e.g., when cloning from spleen cells), follow the instructions for electroporation described in steps 3.3.5.1–3.3.5.3.
7. Plate the transformed cells on 2 x YT, 1% glucose, chloramphenicol (30 µg/ml) 100µg/ml carbenicillin agar plates, and incubate overnight at 37°C.
Note: You may check the ratio of desired ligation product to background by including transformation with “religated” plasmid in the absence of any insert.
Alternatively, the background signal can be analyzed by testing for carbenicillin tetracycline resistance after transformation of other E. coli strains not possessing an intrinsic tet amp resistance (like Invitrogen’s DH5a) with the ligation mix. The portion of vector with unremoved or religated tet cassette is typically in the range of 0.01–0.1%.
3.3.4 Preparation of Electrocompetent E. coli
[edit]
1. For preparation of electrocompetent E. coli XL1-Blue TG1 cells (Stratagene), use 2 ml of a dense overnight pre-culture to inoculate 500 ml medium (2 x YT,
15 µg/ml tetracycline for XL-1 Blue). Shake it at 25°C until an OD600 of 0.6 is reached, then chill the culture on ice as quickly as possible for 30 min (cool the whole shake flask in a large ice bath).
TG1 Genotype: supE thi-1 ∆(lac-proAB) ∆(mcrB-hsdSM)5 (rK – mK – ) [F´ traD36 proAB lacIq Z∆M15].
Note: Sufficient agitation during growth seems to be very important for preparation of electrocompetent cells, reaching reproducible efficiencies of 3–6 x 109 cfu/mg pUC19 DNA. Therefore, use 2 L baffled shake flasks with only 500 ml medium and make sure that the amplitude of the shaker is high enough to vigorously circulate the medium.
Note: The use of electrocompetent bacteria is an alternative to 3.3.3.6 and needed when a large diversity is expected, typically when cloning from spleen cells.
2. Centrifuge the bacterial culture in 50 ml aliquots in disposable tubes for 5 min at 5,000 g. Remove as much supernatant as possible (leave the tube upside down for 15–30 s on a clean tissue). Then, fill each tube with 1 volume of ice-cold distilled water (i.e., the same volume as the original culture aliquot) and remove the water immediately (the cell pellet is very solid after this first centrifugation step and will not be resuspended by the brief rinsing with distilled water).
Note: All these steps should be carried out using ice-cold solutions and be performed in the cold room. Use only ultra pure water to wash cells and to prepare 10% glycerol, as the presence of impurities such as salts in the water might cause the subsequent transformations to fail.
3. Fill each tube with 1 vol distilled water (i.e., the same volume as the original culture aliquot), resuspend the pellet carefully and incubate for 10 min on ice.
Note: Make sure that the cells are sufficiently solubilized to yield a homogeneous suspension. Cells are best resuspended by swirling rather than pipetting. Never vortex the cell suspension!
4. Transfer the cells into new 50 ml tubes and centrifuge at 5,000 g for 10 min. Carefully remove the supernatant and resuspend the pellets each in 50 ml prechilled 10% (v/v) glycerol (Fluka). Incubate on ice for 10 min.
5. Centrifuge resuspended cells at 5,000 g for 15 min and remove the supernatant (you might lose a small portion of cells – do not put the tubes upside down on tissue in this step!). Carefully resuspend the cells in 1/500 of the original culture volume (= 1 ml) 10% (v/v) glycerol, freeze the cells in 100 µl aliquots by dipping the tubes immediately into liquid nitrogen and store them at -80°C.
Note: Electrocompetent cells can be kept at -80°C for up to 12 months.
6. To determine the transformation efficiency, add 1 µl of 10 pg/µl pUC19 DNA (in water) to 40 ml of barely thawed cells (see step 3.3.5.2). Fifty colonies per 1/1,000 of the transformation volume plated correspond to an efficiency of 5 x 109 cfu/µg pUC19 DNA.
3.3.5 Library Preparation/Construction
[edit]1. For desalting the DNA prior to electroporation, apply the ligation mix to StrataClean Resin (Stratagene; hydroxylated silica, binding proteins with a high affinity, while having a low affinity for DNA at near neutral pH), followed by precipitation in 70% ethanol. Since most salts and small organic molecules are soluble in 70% ethanol, they can be separated from DNA by centrifugation.
Resuspend the precipitated DNA in ultra pure water.
2. For each transformation, use desalted ligation mixtures corresponding to 20–100 ng insert. Add the DNA to 40 µl of barely thawed cells on ice and mix by flipping the tube shortly and gently. Immediately transfer the cell-DNA mix to chilled electroporation cuvette (bubble free), pulse according to the guidelines of the electroporator’s manufacturer, and add 1 ml of SOC medium (20 g/l bacto-tryptone, 5 g/l yeast extract, 10 mM NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgCl2, 10 mM MgSO4) to cells immediately after the pulse.
Note: For efficient transformation ( ≥ 108 clones per µg insert DNA), the time constant using 2 mm cuvettes should be ≥ 5 ms, reflecting properly washed cells.
Also, make sure that no air-bubbles are trapped in the cell-DNA mix as they will interfere with the electroporation.
3. Resuspend cells completely in SOC medium and shake for 1 h at 37°C. Afterward, plate dilutions on 2 x YT, 1% glucose, 100µg/ml carbenicillin chloramphenicol (30 µg/ml) agar plates.
Use a sterile spreader or sterile glass beads to evenly distribute the culture over the surface of the 12 x 12 cm plate (do not exceed about 5,000 clones per square plate) and incubate overnight at 37°C. The next day, scrape the colonies off the plates in 3–4 ml 2 x YT, containing 30% glycerol, and subsequently store them at -80°C.
Note: Take care that your library is homogeneously mixed.
4. For phage panning as described in 3.3.6, inoculate cultures with at least tenfold more viable cells than colonies obtained after transformation, in order to have sufficient oversampling. When starting from spleen cells, perform three rounds of phage panning as described in 3.3.6 before testing single clones. When starting from a hybridoma, one round should be sufficient, and in ideal cases, single clones can be tested right away by phage ELISA.
Note: The first panning round is the most crucial, as you might lose any desired, but less abundant antibody sequence by too extensive washing. Therefore, do not exceed ten washing steps in this first panning round. The panning procedure is analogous to the phage ELISA (3.3.6), except that a pool of phages is grown and that phages are eluted from antigen (at the end of 3.3.6.4), which are afterward added again to exponentially growing bacteria. This is described in detail elsewhere (Barbas et al. 2001; Lee et al. 2007) and also in this volume.
Note: The screening of single clones can be performed in three ways. First, at the level of phages (phage ELISA), as described in Sect. 3.3.3.6, second, after retransforming of an suppressor tRNA-deficient strain such as, e.g., E. coli strain JM83 (Yanisch-Perron et al. 1985), still with the amber codon, containing pAK100 derived plasmids, or third, after recloning into a more efficient expression vector such as pAK400 (Fig. 3.5), which carries no gene III. In second and third option, the soluble scFv is screened by ELISA.
Note: This protocol does not describe the periplasmic expression of scFv fragments in E. coli (Glockshuber et al. 1990) and their subsequent purification (reviewed in Plu¨ckthun et al. 1996). More details can be found in chapter 27 “Improving expression of scFv fragments by coexpression of periplasmic chaperones” in this volume.
Another Protocol
[edit]ELECTROPORATION PROTOCOL
1. Pre-chill two sterile electroporation cuvettes ('''0.1-cm''' gap) and two sterile 1.5-ml microcentrifuge tubes thoroughly on ice. Preheat sterile
SOC medium to 37°C.
2. Set the electroporator to a voltage setting of '''1700 V''' (17 kV/cm field strength). If using a Bio-Rad electroporator, set the resistance at '''200 Ω''' and the '''capacitance at 25 µF'''. For best results, chill the electroporator or perform the electroporation in a cold room.
3. Thaw the electroporation-competent cells on ice. After mixing the cells gently, aliquot '''40 µl''' of cells into each of the two pre-chilled tubes (one tube for the experimental transformation and one tube for the pUC18 control transformation). Keep the tubes on ice.
4. Add the DNA to the cells with gentle mixing. For optimal efficiency in the experimental transformation, add '''1 µl''' of plasmid DNA ('''10 pg/µl''', in a low ionic strength buffer or dH2O) to '''40 µl''' of cells. '''The DNA volume may be increased up to 4 µl''', but the efficiency may be reduced. Dilute the pUC18 control DNA 1:10 with sterile dH2O, then add 1 µl of the diluted pUC18 DNA to the other 40 µl of cells.
5. Transfer the cell-DNA mixture to a chilled electroporation cuvette, tapping the cuvette until the mixture settles evenly to the bottom.
6. Slide the cuvette into the electroporation chamber until the cuvette sits flush against the electrical contacts.
7. Pulse the sample once, then quickly remove the cuvette. '''Immediately add 960 µl of SOC medium (held at 37°C) to resuspend the cells.'''
8. Transfer the cells to a sterile 14-ml BD Falcon polypropylene round-bottom tube (BD Biosciences Catalog #352059). Incubate the tube at 37°C for 1 hour with shaking at 225–250 rpm.
9. Plate 5–100 µl of the transformation mixture on LB agar plates containing the appropriate antibiotic (and containing IPTG and X-gal if color screening is desired).§
For the pUC18 control transformation, plate 2.5 µl of the transformation on LB-ampicillin agar plates.§
Notes If plating <100 µl of cells, pipet the cells into a 200 µl pool of SOC medium and then spread the mixture with a sterile spreader.
If plating ≥100 µl, the cells can be spread on the plates directly. Tilt and tap the spreader to remove the last drop of cells.
Some β-galactosidase fusion proteins are toxic to the host bacteria. If an insert is suspected to be toxic, plate the cells on media without X-gal and IPTG. Color screening will be eliminated, but lower levels of the potentially toxic protein will be expressed. 10. Incubate the plates at 37°C overnight (less than 24 hours, and at least 17 hours for blue-white color screening). If blue-white screening was performed, colonies containing plasmids with inserts will remain white, while colonies containing plasmids without inserts will be blue. The blue color can be enhanced by incubating the plates for two hours at 4°C following the overnight incubation at 37°C. 11. For the pUC18 control, expect 250 colonies (≥1 × 1010 cfu/µg pUC18 DNA). For the experimental DNA, the number of colonies will vary according to the size and form of the transforming DNA, with larger and non-supercoiled DNA producing fewer colonies.
3.3.6 Screening for Binders by Phage ELISA
[edit]
1. When starting from hybridoma, pick 10 colonies (from spleen, as many as you can handle) and grow them separately at 37°C in 2 ml 2 x YT, 1% glucose, 100µg/ml carbenicillin chloramphenicol (30 µg/ml) until they reach an OD600 of 0.5. This level of glucose fully represses expression, and, thus, the growth temperature can be 37°C.Dilute 1:10 in 2 x YT, 1% glucose, 100µg/ml carbenicillin chloramphenicol (30 µg/ml), containing 1 mM IPTG, and 1 x 1010 pfu VCSM13 helper phage (Stratagene) per ml, and grow overnight at 26°C or 37°C (for some murine scFvs with aggregation tendencies, growth at 26°C after infection may be necessary). The phage titer after overnight incubation is in the range of 1011–1012 cfu per ml supernatant.
Note: XL1-Blue should be grown on agar plates and in media containing tetracycline (tet) as the F0-plasmid encoding for the F-pili required for infection
of bacteria also carries the tet resistance gene. The phage titer (in cfu) should be determined in order to rule out any problems during phage production. To do so, take a log-phase culture of XL1-Blue cells (OD600 = 0.4–0.6) and incubate aliquots of this culture with serial dilutions of your phage preparation. After 15 min incubation at 37°C, plate appropriate amounts (30–150 cfu/plate) on 2 x YT, 1% glucose, chloramphenicol (30 µg/ml) agar plates.
Note: We are aware of the fact that the presence of such a high level of glucose during IPTG-induction is rather unusual. However, for phage display, the induction level does not have to be very high. Based on our experiences with this vector series described here, a combination of IPTG addition and the presence of glucose seems to be crucial for the successful expression of some scFv fragments, notably those with nonideal biophysical properties, and appropriate for most, but may have to be checked for each scFv individually in case of
unusual properties.
2. Centrifuge the culture 10 min at 16,000 g and 4°C. Take 1.6 ml supernatant and mix it with 0.4 ml 20% PEG 6000 (Fluka), 2.5 M NaCl in a 2 ml Eppendorf tube in order to precipitate the phages (Sambrook and Russell 2001).
Note: We recommend that the PEG solution be freshly prepared.
3. Incubate on ice for 30–60 min and centrifuge for 15 min at 5,600 g and 4°C.
Note: It is important not to centrifuge phages at too high a g force, as otherwise, it will be difficult to resuspend them homogeneously, resulting in a decreased phage titer. The size of the white pellet does not necessarily reflect a high or low phage titer.
4. Resuspend the phage pellet in 400 µl PBS (with 10% (v/v) glycerol). For complete resuspension, incubate the phage solution on an orbital shaker at 800 rpm for 15 min at 4°C. Pellet insoluble matter (cell debris) by centrifugation for 10 min at 11,000 g and 4°C and transfer the phage solution to a fresh tube.
Use 100 µl phage solution per well in an ELISA assay to distinguish phages displaying functional scFv antibody from those which display nonfunctional or nonproductive antibody fragments.
5. If soluble antigen is available, include a competition ELISA control showing that free antigen is able to compete with bound antigen for phage binding to distinguish nonspecific “sticky” from specifically binding phages. In principle, the same ELISA protocol that was used for the hybridoma screening procedure can be used.
Note: For weak binders, it might be important to use more phages for ELISA analysis. In this case, the culture volume should be increased ten times. If no functional clone shows up in ELISA of single clones, perform one round of phage panning in order to enrich the functional binders. The enrichment should be checked by comparison of eluted phages from a specific surface versus a surface without antigen. In addition, it is recommended to analyze the phage solution by immunoblot, using an anti-M13 HRP-conjugated antibody (GE
Healthcare), to ensure the correct fusion of the scFv to the gIII-protein as well as its correct display on the phage surface.
3.4 Troubleshooting
[edit]
This part of the protocol contains general comment about potential pitfalls of the recommended standard method. The most critical steps were already highlighted
directly following the instructions in the different subsections.
(a) In case of low transformation yields, check whether the problem is the transformation itself or rather the ligation. To investigate the quality of ligation, analyzing an aliquot by agarose gel electrophoresis might indicate any problems caused by nucleases. Furthermore, it might be informative to compare the ligation efficiency of SfiI digested PCR product with inserts derived from plasmid digestion. In order to check both the ligation and the transformation efficiency, a defined amount of pUC19 DNA can be added to the ligation mixture. Because of the chloramphenicol resistance of the cloning vector and the ampicillin resistance of pUC19 DNA, it is possible to calculate the ligation efficiency by plating double transformed cells on ampicillin or chloramphenicol plates, respectively, and comparing the number of clones. The transformation efficiency (in presence of the ligation mixture) can be judged by comparison of the colony number after transformation with pUC19 DNA alone
(b) The quality of the oligonucleotides used in this procedure is crucial for the successful and reliable amplification of various antibody genes as well as their subsequent assembly into scFvs. The number of proposed primers is important for a broad representation of the immune response, as any sequence absent from the complex mixture will obviously decrease the functional library size. We also strongly recommend using primers that have been accurately purified after their synthesis (either by HPLC or, for longer primers, by PAGE) to ensure that
no single-base deletions are present in any of the oligonucleotides. These deletions as well as any insertions would cause frameshifts in the final gene assembly, resulting in a number of nonfunctional library members. Therefore, we also suggest – especially for library cloning – to sequence the genes of several random clones as well as to check for full-length scFv by western blot analysis detecting its fusion partner gene III (see note at 3.3.6.5).
(c) In case of severe problems in the PCR amplification of the VH and VL genes (steps 3.3.2), it might be worth considering to divide this reaction into two separate ones. Using the proposed primers without any overhang at their 50-end (which either codes for the FLAGs-tag and the SfiI cleavage site, or the (Gly4Ser)4-linker), the pure antibody DNA should be amplified in a first PCR reaction, and, subsequently, a second PCR should be performed for reamplification and introduction of the appropriate overhangs with the original
full-length primers. This procedure increases the degree of matching in both reactions, and might therefore help in the annealing step of the primers.
(d) Whenever expression of the scFv gene is not required, the bacteria should be grown in the presence of 1% glucose. Glucose will cause a tight suppression of
the lac promoter, thereby ensuring the genetic stability of the inserted scFv genes. Likewise, we suggest growing XL1-Blue always on agar plates and in media containing tetracycline (tet) to keep the bacteria infective, as the tet resistance is located on the F0-plasmid that also contains the genes encoding F-pilus formation. Always use fresh XL1-Blue colonies, as subcloning might occasionally lead to the formation of tet resistant cells, which are no longer infectable. As the F-pilus expression is reduced when the bacteria are past log phase as well as when grown at temperatures below 34°C, we also recommend growing them at 37°C to OD600 = 0.4–0.6.
(e) When working with libraries, double transformants can and will occur (Goldsmith et al. 2007). It is thus highly recommended that the scFv fragments of interest be recloned into a new vector (e.g., from pAK100 to pAK400), when they are analyzed at the level of pure unfused protein, thereby also introducing a stronger translation initiation region. It should be noted that diluted retransformation cannot resolve plasmid mixtures, as in E. coli, plasmids can form reversible concatamers.