This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.
Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.
mAbs have broad applications ranging from basic research to disease diagnostics and therapeutics. As of 2016, more than 60 mAbs have been approved by the United States Food and Drug Administration (USFDA) for clinical treatment of autoimmune diseases, cancer, and infectious diseases1,2.
In 1975, Kohler and Milstein reported a technique for the continuous generation of antibodies of a single clonal specificity from a cellular source referred to as 'hybridomas' and this technique has subsequently become a cornerstone in medicine and industry3,4. Generation of mAbs by this method requires various steps including antigen production, mouse immunization, extraction of B lymphocytes, fusion of B cells with myeloma cells to form immortal hybridoma cells, clone selection, and for therapeutic applications, humanization is required to avoid human anti-mouse antibody (HAMA)4,5. However, for this technology, antigens including toxins, pathogens, and highly conserved proteins are relatively ineffective in triggering an in vivo immune response for mAb production5.
In 1978, Hutchison et al. reported the use of an oligonucleotide to direct mutagenesis of a residue in a single-stranded bacteriophage virus6. In 1985, Smith reported that foreign gene fragments can be fused in frame with the gene encoding phage coat protein III and can thus be displayed on the phage surface without compromising its infectivity7. These pioneering works laid a foundation for the subsequent construction of phage-displayed antibody libraries in immune, naïve, and synthetic forms with the formats of single-chain variable fragment (scFv) and antigen-binding fragment (Fab) for therapeutic mAb development8,9. From the technical point of view, phage display-based antibody development offers a complementary approach to hybridoma-based mAb development that can help to circumvent the limitations some antigens can pose and the humanization process that hybridoma-derived antibodies often require5. As of 2016, 6 phage display-derived mAbs have been approved in the market including Humira, one of the most successful mAbs used for treatment of rheumatoid arthritis, and many phage display-derived antibody candidates are currently at various stages of clinical investigation10.
For immune and naïve phage antibody libraries, the diversity of complementarity-determining regions (CDRs) in light and heavy chain is derived from the natural immune repertoire (i.e., from B cells). In contrast, the diversity of CDRs in synthetic phage antibody libraries is entirely artificial. Synthetic approaches to library construction provide precise control over the design of sequence diversity and offer opportunities for mechanistic studies of antibody structure and function11,12. Moreover, the framework for synthetic libraries can be optimized before library construction to facilitate downstream, large-scale industrial development11,12.
In 1985, Kunkel reported a single-stranded DNA (ssDNA) template-based mutagenesis approach to introduce site-directed mutations into M13 bacteriophage efficiently13. This approach was subsequently used widely for construction of phage-displayed libraries. Chemically synthesized DNA oligonucleotides designed to introduce diversity into Fab CDRs are incorporated into a phagemid with an antibody backbone template. In this process, the phagemid is expressed as a uracil-containing ssDNA (dU-ssDNA) and the oligonucleotides are annealed onto the CDRs and extended to synthesize double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase and T4 DNA ligase. Finally, generated ds-DNA can be introduced into Escherichia coli by electroporation.
For high diversity, phage-displayed library construction, high-voltage electroporation of a two-component mixture of electro-competent cells and covalently closed circular dsDNA (CCC-dsDNA) should be prepared carefully. Sidhu et al. modified the preparation of electro-competent cells and DNA from traditional methods and greatly improved library diversity14.
In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. Figure 1 shows an overview of library construction including: 1) high-efficiency electro-competent cell preparation; 2) extraction of dU-ssDNA; 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based ELISA for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity. All the reagents, strains and equipment are listed in the Material's Table. Table 1 shows the reagent setup.
NOTE: Filter sterile tips must be used throughout when dealing with phage to avoid contamination to pipette gun and surrounding area. Aseptic area or hood must be used when handling with bacteria and phage experiments. Phage experiment area must be cleaned up using 2% sodium dodecyl sulfate (SDS) followed by 70% ethanol to avoid phage contamination. For making serial dilutions in this protocol, new tips should be used for each dilution.
1. E. Coli SS320 Electro-competent Cell Preparation
2. Preparing Uracil-containing ssDNA (dU-ssDNA) from the Phagemid Template
NOTE: A previously reported Fab backbone phagemid was used as the template for dU-ssDNA preparation15. The architecture of the Fab backbone phagemid is shown in Figure 2. A plasmid spin kit (QIAprep Spin M13) is used for extraction of dU-ssDNA with slight modifications.
3. Kunkel's Method Based Oligonucleotide-directed Mutagenesis
Notes: It is advisable to conduct small-scale reactions prior to full scale reactions to ensure the quality of the mutagenic oligonucleotide and reaction components. A cartoon of Kunkel's method based oligonucleotide-directed mutagenesis is shown in Figure 3. Various amino acid diversities are introduced into CDRH1, CDRH2, CDRH3, and CDRL3 regions with IMGT numbering nomenclature16 (Table 2). The theoretical amino acid diversity of each CDR, total theoretical amino acid diversity, and oligonucleotide sequences are listed in Table 2.
4. Electroporation and Calculation of the Library Size
5. Quality Assessment by Protein A/L Direct Binding ELISA Assay and Sequencing
Following the flow chart of the Fab library construction (see Figure 1), we prepared M13KO7 helper phage pre-infected E. coli SS320 electro-competent cells. The efficiency of these electro-competent cells is estimated as 2 X 109 cfu/µg when the Fab phagemid backbone for library construction was used (Figure 4).
The uracil incorporation efficiency by comparison of titer in both E. coli CJ236 and E. coli SS320 cells was checked. The E. coli CJ236 and E. coli SS320 cells were infected by phage harboring dU-ssDNA. E. coli SS320 has enzymes (dUTPase and uracil glycosylase) that can degrade uracil-containing DNA, while E. coli CJ236 lacks these enzymes and cannot degrade uracil-containing DNA. To achieve an acceptable uracil incorporation efficiency, titers from E. coli CJ236 need to be at least 104 times higher than those from E. coli SS320. Otherwise the wild-type population will increase in the constructed antibody library due to inefficient uracil incorporation. Figure 5 showed that the titer from E. coli CJ236 is approximately 3 X 105 times higher than that from E. coli SS320, indicating an efficient uracil incorporation into phage ssDNA.
Next, we prepared and extracted dU-ssDNA. The dU-ssDNA purity is checked by agarose gel electrophoresis (Figure 6). Then the oligonucleotide-directed mutagenesis was conducted and the efficiency of the dU-ssDNA conversion to CCC-DNA was evaluated (Figure 6). Three products with lower motility than dU-ssDNA can be visualized on the gel including the fastest-running band (CCC-dsDNA), the middle weak band (nicked band), and the slowest-running band (strand-displaced DNA).
After electroporation into E. coli SS320, the library size was estimated from the overnight incubation plate (see step 4.7.5). The average library size was 5 X 109 from duplicate serial dilutions on LB/carb plates (Figure 7). However, the estimated size at this step may contain phage that do not display Fabs due to the presence of a frameshift or stop codon, or display misfolded Fabs. Sequencing and ELISA were used to estimate the functional diversity of constructed library. 96 randomly picked single clones were sent for sequencing analysis. Table 4 shows that 90 out of the 96 randomly picked single clones were successfully sequenced, which contains 70 clones without a premature stop codon (53 clones with at least one CDR mutant and 17 clones with the wild-type sequence) and 20 clones with a premature stop codon at different regions. Within the 70 clones, mutant rates of CDRH1, CDRH2, CDRH3, and CDRL3 are 50%, 57%, 53%, and 56%, respectively, while the mutant rate with at least one CDR is 76%. In the 20 clones with a premature stop codon (90%), the premature stop codon was mainly derived from the frameshift of oligonucleotide mutagenesis primers, including 45% (CDRH1), 10% (CDRH2), 15% (CDRH3), and 20% (CDRL3).
To detect the display of properly folded Fabs, a protein A/L based ELISA was employed as it is known that protein A and protein L can recognize proper folding of the VH framework and VL framework, respectively17,18. In agreement with the sequencing analysis, the ELISA assay in triplicate (Figure 8) showed that the 20 clones with a premature stop codon were all negative while the 17 clones with a wild-type sequence were all positive when the positive ratio was empirically set at 3.0. For the 53 clones with at least one CDR mutant, 43 clones were positive in ELISA while 10 clones were negative; this indicates that most of the clones were well folded while the CDRs from the 10 clones can have detrimental effects on Fab folding. In total, 43 clones out of the 90 clones (48%) were well folded and contained at least one CDR mutant. Thus, the functional amino acid diversity of the constructed library based on protein A/L ELISA and sequence analysis was estimated to be 2.4 X 109 (i.e., 48% of 5 X 109).
Figure 1: Overview of the phage-displayed Fab library construction. Phage-displayed Fab library construction follows a basic series of steps. It involves preparation of high-efficiency electro-competent bacterial cells, extraction of dU-ssDNA, Kunkel's method based oligonucleotide-directed mutagenesis, electroporation and calculation of phage Fab library size, functional evaluation by protein A/L ELISA, and DNA sequence analysis. Please click here to view a larger version of this figure.
Figure 2: Phagemid architecture for the Fab library construction. The basic features of the phagemid backbone consist of origins of single-stranded (f1 ori) and double-stranded (dsDNA ori) DNA replication, and an ampicillin/carbenicillin resistance gene (AmpR). For Fab display, under the control of the alkaline phosphatase promoter (PhoA), the phagemid contains a bicistronic cassette to drive expression and secretion of: light chain (LC) consisting of a secretion signal, VL (variable region of light chain), CL (constant region of light chain), and C-terminal flag tag; and heavy chain (HC) consisting of a secretion signal, VH (variable region of heavy chain), and CH1 (constant region 1 of heavy chain) fused with a p3 phage minor coat protein. Assembly of the light chain and heavy chain into Fab within the E. coli periplasm directs the display of Fab on the phage surface. Please click here to view a larger version of this figure.
Figure 3: Schematic of Kunkel's method based oligonucleotide-directed mutagenesis. In this protocol, we used Kunkel's method to prepare dU-ssDNA template. Oligonucleotides for CDRH1, CDRH2, CDRH3, and CDRL3 with designed diversity are phosphorylated, annealed to the template, and used to convert ss-DNA to CCC-dsDNA. Following electroporation into E. coli SS320 electro-competent cells, the heteroduplex DNA is repaired to either the wild type or the mutant form; Due to the presence of uracil in the wild type strand, the repair process favors the mutant form, and thus, the mutant form dominates the library. Please click here to view a larger version of this figure.
Figure 4: Estimation of the M13KO7 pre-infected E. coli SS320 electro-competent cell efficiency. A phagemid backbone vector was used to check the electroporation efficiency of the competent cells. Formula to calculate the efficiency is as follows: assume that M is the average colony number counted from the most diluted fold 10N (N is from 1-8) in duplicate. E. coli SS320 efficiency from LB/carb plate is equal to M X 10N+3 cfu/µg. The efficiency of the electro-competent cells is around 2 X 109 cfu/µg. Please click here to view a larger version of this figure.
Figure 5: Assessment of uracil incorporation into ssDNA by phage infection of E. coli CJ236 and E. coli SS320 cells. Based on Kunkel's method, the uracil incorporation efficiency is checked by comparison of phage infection titer in both E. coli CJ236 and E. coli SS320 cells. The titer calculation formula is as follows: assume that M is the average colony number counted from the most diluted fold 10N (N is from 1-10), and that the titer from E. coli CJ236 or E. coli SS320 is equal to M X 10N+2 cfu/mL. The efficiency of uracil incorporation can be estimated from the titer ratio of E. coli CJ236 and E. coli SS320. The titer in E. coli CJ236 was 9 X 1012 cfu/mL while the titer in E. coli SS320 was 3 X 107 cfu/mL. The titer ratio of E. coli CJ236 and E. coli SS320 was 3 X 105. Please click here to view a larger version of this figure.
Figure 6: Conversion of dU-ssDNA to CCC-DNA by oligonucleotide-directed mutagenesis. Following oligonucleotide-directed mutagenesis, the efficiency of dU-ssDNA conversion to CCC-DNA was evaluated. dU-ssDNA was completely converted to dsDNA. The dominant band is CCC-dsDNA while there is a minor portion of nicked dsDNA and strand-displaced DNA. Please click here to view a larger version of this figure.
Figure 7: Phage titration for calculation of library size. After electroporation into E. coli SS320, the library size was estimated from serial dilutions on LB/carb plates. The size calculation formula is as follows: assume M is the average colony number counted from the most diluted fold 10N from a 2YT/Carb plate (N is from 1-8), size is equal to 2M X 10N+3. Please click here to view a larger version of this figure.
Figure 8: Protein A/L direct binding phage ELISA. Protein L can recognize the framework of well folded kappa light chain VL and protein A can recognize the framework of well folded heavy chain VH. Binding of Fab with protein L and A indicates proper folding of both heavy chain and light chain. In brief, protein L in triplicate and the negative control M-PBST were coated to the plate, Fab phage supernatants from different clones were incubated with protein L and M-PBST, then after wash, protein A-HRP was used to capture bound Fab phage. Phage ELISA readings showed 90 randomly picked clones with successful sequencing readout. A threshold line representing the clone as positive was empirically defined where the ratio of OD450 absorbance value from protein L (average of triplicate with error bar) versus negative control was more than 3.0. Three groups based on sequencing analysis were shown, corresponding to a mutant without stop codon in red (53 clones), wild type (WT) in blue (17 clones), and mutant with stop codon in green (20 clones). Please click here to view a larger version of this figure.
Reagent setup | Component | Amount | comments/description |
2YT medium | Yeast extract | 10 g | Add ultrapure water to make up the volume to 1.0 L, adjust pH to 7.0, autoclave. |
Tryptone | 16 g | ||
NaCl | 5 g | ||
2YT top agar | Yeast extract | 10 g | Add ultrapure water to make up the volume to 1.0 L and adjust pH to 7.0, heat to dissolve, autoclave. |
Tryptone | 16 g | ||
NaCl | 5 g | ||
Granulated agar | 7.5 g | ||
2YT/carb/cmp medium | Carbenicillin | 100 μg/mL | |
Chloramphenicol | 10 μg/mL | ||
2YT/carb/kan/uridine medium | Carbenicillin | 100 μg/mL | |
Kanamycin | 50 μg/mL | ||
Uridine | 0.25 μg/mL | ||
2YT/carb/tet medium | Carbenicillin | 100 μg/mL | |
Tetracycline | 10 μg/mL | ||
2YT/carb medium | Carbenicilin | 100 μg/mL | |
2YT/kan medium | Kanamycin | 50 μg/mL | |
2YT/kan/tet medium | Kanamycin | 50 μg/mL | |
Tetracycline | 10 μg/mL | ||
2YT/tet medium | Tetracycline | 10 μg/mL | |
2YT/cmp medium | Chloramphenicol | 10 μg/mL | |
LB medium agar | Yeast extract | 5 g | Add ultrapure water to make up the volume to 1.0 L, adjust pH to 7.0, autoclave. For LB agar, add 20 g of granulated agar, autoclave. |
Tryptone | 10 g | ||
NaCl | 10 g | ||
LB/carb plates | LB agar | 1L | |
Carbenicillin | 100 μg/mL | ||
LB/tet plates | LB agar | 1 L | |
Tetracycline | 10 μg/mL | ||
LB/cmp plates | Chloramphenicol | 10 μg/mL | |
LB/kan plates | Kanamycin | 50 μg/mL | |
SOC medium | Yeast extract | 5 g | Add ultrapure water to make up the volume to 1.0 L and adjust pH to 7.0, autoclave. |
Tryptone | 20 g | ||
NaCl | 0.5 g | ||
KCl | 0.2 g | ||
2.0 M MgCl2 | 5.0 mL | ||
1.0 M glucose | 20 mL | ||
Superbroth medium | Tryptone | 12 g | Add ultrapure water to 900 mL, autoclave, add 100 mL of autoclaved 0.17 M KH2PO4, 0.72 M K2HPO4. |
Yeast extract | 24 g | ||
Glycerol | 5 mL | ||
Superbroth kan/tet medium | Kanamycin | 50 μg/mL | |
Tetracycline | 10 μg/mL | ||
1X PBS | NaCl | 137 mM | Adjust pH to 7.2, autoclave. |
KCl | 3 mM | ||
Na2HPO4 | 8 mM | ||
KH2PO4 | 1.5 mM | ||
TAE/agarose gel | TAE buffer | ||
Agarose | 1% (w/v) | ||
GelRed | 1:10000 (v/v) | ||
TMB substrate | TMB | 50% (v/v) | |
H2O2 peroxidase substrate | 50% (v/v) | ||
M-PBST buffer | 1X PBS | 100 ml | |
Tween-20 | 0.05% (v/v) | ||
NON-Fat Powdered Milk | 5% (v/v) | ||
5X PEG/NaCl | PEG-8000 | 20% (w/v) | Add ultrapure water to make up the volume to 1L, and autoclave. |
NaCl | 2.5 M | ||
PBST buffer | 1X PBS | 1 L | 0.22 μm filter-sterilize. |
Tween-20 | 0.05% (v/v) | ||
10X TM buffer | MgCl2 | 0.1 M | Adjust pH to 7.5. |
Tris | 0.5 M | ||
1.0 mM HEPES, pH 7.4 | 1.0 M HEPES | 4.0 mL | 0.22 μm filter-sterilize. |
Ultrapure water | 4.0 L | ||
10% (v/v) ultrapure glycerol | Ultrapure glycerol | 100 ml | 0.22 μm filter-sterilize. |
Ultrapure water | 900 mL | ||
Ultrapure water | H20 | Dnase-free, Rnase-free, Pyrogen-free. |
Table 1: Reagent setup.
Table 2: CDR diversities and mutagenesis primers. The DNA sequences of the CDR regions to be mutated are shown in red; sequences are formatted using the IUPAC nucleotide code. "X" indicates tri-nucleotide from a mixture designed to contain different amino acid sets; "n" indicates different number of X. Five primers with a different number of X were used to diversify CDRL3 or CDRH3, respectively, to generate variable length of CDRL3 and CDRH3. The residue numbers are defined by the IMGT nomenclature. Please click here to download this table.
Kunkel's method based mutagenesis | ||
Reaction 1. Oligonucleotide phosphorylation with T4 polynucleotide kinase | ||
Component | Amount | Final |
mutagenic oligonucleotides | 0.6 μg | |
10X TM buffer | 2 μL | 1X |
10 mM ATP | 2 μL | 1 mM |
100 mM DTT | 1 μL | 5 mM |
T4 polynucleotide kinase (10 U/μL) | 2 μL | 20 U |
Ultrapure H20 | Up to 20 μL | |
Reaction setting | ||
Step 1. | 37 °C for 1 h | |
Reaction 2. Annealing of the oligonucleotides to the template | ||
Component | Amount | Final |
dU-ssDNA template | 20 μg | 20 μg |
10X TM buffer | 25 μL | 1X |
phosphorylated CDRH1 oligonucleotides | 20 μL | 0.6 μg |
phosphorylated CDRH2 oligonucleotides | 20 μL | 0.6 μg |
phosphorylated CDRH3 oligonucleotides | 20 μL | 0.6 μg |
phosphorylated CDRL3 oligonucleotides | 20 μL | 0.6 μg |
Ultrapure H20 | Up to 250 μL | |
Reaction setting | ||
Step 1. | 90 °C for 3 min | |
Step 2. | 50 °C for 5 min | |
Step 3. | 20 °C for 5 min | |
Reaction 3. Enzymatic synthesis of CCC-dsDNA | ||
Component | Amount | Final |
annealed oligonucleotides/template mixtures | 250 μL | |
10 mM ATP | 10 μL | 346 µM of each nucleotide |
dNTP mix (25 mM of each nucleotide) | 10 μL | 865 µM of each nucleotide |
100 mM DTT | 15 μL | 5 mM |
T4 DNA ligase | 1 μL | 30 Weiss units |
T7 DNA polymerase | 3 μL | 30 U |
Reaction setting | ||
Step 1. | 20 °C for overnight |
Table 3: Procedures and components of Kunkel's method based reaction.
Group | Clone number | Region | Percentage | ||
No premature stop codon | 70 | CDRH1 mutation | 50% (35/70) | ||
CDRH2 mutation | 57% (40/70) | ||||
CDRH3 mutation | 53% (37/70) | ||||
CDRL3 mutation | 56% (39/70) | ||||
At least one CDR mutation | 76% (53/70) | ||||
Premature stop codon | 20 | CDRH1 defect | 45% (9/20) | ||
CDRH2 defect | 10% (2/20) | ||||
CDRH3 defect | 15% (3/20) | ||||
CDRL3 defect | 20% (4/20) | ||||
Other defect | 10% (2/20) |
Table 4: Sequence analysis of CDRH1, CDRH2, CDRH3, and CDRL3 from the synthetic Fab library.
To construct high diversity, phage-displayed Fab libraries, quality control check points are needed to monitor various stages of the construction process, including the competency of electro-competent cells, quality of the dU-ssDNA template, efficiency of CCC-dsDNA synthesis, titer after electroporation, Fab folding, and amino acid diversity of CDRs by sequence analysis of Fab-phage clones.
High yield and purity of dU-ssDNA is essential for high mutagenesis rate. In our experience, phage induction at 25 °C overnight can yield more dU-ssDNA than that from phage induction at 37 °C overnight. This is in agreement with a previous report19. Regarding ssDNA extraction, the initial plasmid Spin kit (QIAprep) contained MLB for phage lysis and binding. Later, MLB was discontinued with unknown reason and replaced by PB. We found that the yield of dU-ssDNA is much lower from PB treatment as compared with that from MLB treatment. In this protocol, we used a reagent named UT-MLB20 to replace MLB and found the yield of dU-ssDNA is similar to that from the initial Spin Kit.
As CDRH3 and CDRL3 are the most diverse regions for antigen recognition21, to introduce a tailored diversity with a specific set of amino acid combinations and ratios, and to remove redundancy bias and stop-codons introduced by degenerate codons such as NNK (N, equimolar of A/C/G/T; K, equimolar of G/T), trimer codon phosphoramidite-based oligonucleotides22 with exactly one trimer codon corresponding to one specific amino acid were designed for CDRH3 and CDRL3. Moreover, variable lengths of CDRH3 and CDRL3 oligonucleotides were used to further increase diversities.
After enzymatic synthesis of CCC-dsDNA, generally three bands are observed by agarose gel electrophoresis and the bands should be clear without smear. Among them, the fastest-running band is the CCC-dsDNA that can yield a high mutation rate (around 80%) after electroporation23. The slowest-running band is the strand-displaced DNA that arises from inherent strand-displacement activity of T7 DNA polymerase and has a low mutation rate (around 20%)23. The middle weak band is nicked DNA after extension due to insufficient T4 DNA ligase activity or insufficient oligonucleotide phosphorylation.
A small sequencing sample pool was used to estimate the library diversity though not accurate24. To estimate the real diversity accurately, next generation sequencing (NGS) may be a good option in mining the diversity depth of the constructed library25. In practice, due to the current challenges of NGS technology including read length, accuracy, cost, and high throughput, the sequencing of the Fab phage library used in this protocol with the length of around 950 bp spanning CDRH1, CDRH2, CDRH3, and CDRL3 is not achievable; however, it is possible to estimate the scFv (around 700 bp) library diversity within the range of millions24,25. Another key standard to evaluate the diversity of constructed library is to use the library to pan against many different types of antigens and calculate the positive clones captured since library diversity is directly correlated with the successful rate of antigen panning26. High throughput selection platform is well suited for this purpose and readers can refer to a detailed protocol reported by Miersch et al.27
Theoretically, phage-displayed synthetic antibody libraries with tailored diversity can be used to target any antigen and thus have broad applications. Currently, companies including Cambridge Antibody Technology (CAT), MedImmune, Genentech, Dyax, Bioinvent, Pfizer, and MorphoSys rely heavily on phage display platforms for therapeutic antibody development28. Moreover, many phage display core technology patents have expired29. Undoubtedly, this will unleash the maximum potential of phage-displayed antibody technology.
The authors have nothing to disclose.
The authors appreciate Dr. Frederic Fellouse from the Sidhu lab for critical comments on Kunkel's method based synthetic Fab phage library construction. The authors appreciate Mrs. Alevtina Pavlenco and other members from the Sidhu lab for valuable help of preparing high-efficiency electro-competent E. coli cells and high quality dU-ssDNA. This work was supported by National Natural Science Foundation of China (Grant No.: 81572698, 31771006) to DW and by ShanghaiTech University (Grant No.: F-0301-13-005) to Laboratory of Antibody Engineering.
Reagents | |||
1.0 M H3PO4 | Fisher | AC29570 | |
1.0 M Tris, pH 8.0 | Invitrogen | 15568-025 | |
10 mM ATP | Invitrogen | 18330-019 | |
100 mM dithiothreitol | Fisher | BP172 | |
100 mM dNTP mix | GE Healthcare | 28-4065-60 | solution containing 25 mM each of dATP, dCTP, dGTP and dTTP. |
3,3’,5,5’-tetramethylbenzidine (TMB) | Kirkegaard & Perry Laboratories Inc | 50-76-02 | |
50X TAE | Invitrogen | 24710030 | |
Agarose | Fisher | BP160 | |
Carbenicillin, carb | Sigma | C1389 | 100 mg/mL in water, 0.22 μm filter-sterilize, work concentration: 100 μg/mL. |
Chloramphenicol, cmp | Sigma | C0378 | 100 mg/mL in ethanol, 0.22 μm filter-sterilize, work concentration: 10 μg/mL. |
EDTA 0.5 M, pH 8.0 | Invitrogen | AM9620G | |
Granulated agar | VWR | J637-500G | |
H2O2 peroxidase substrate | Kirkegaard & Perry Laboratories Inc | 50-65-02 | |
K2HPO4 | Sigma | 795488 | |
Kanamycin, kan | Fisher | AC61129 | 50 mg/mL in water, 0.22 μm filter-sterilize, work concentration: 50 μg/mL. |
KH2PO4 | Sigma | P2222 | |
Na2HPO4 | Sigma | 94046 | |
NaCl | Alfa Aesar | U19C015 | |
Nanodrop | Fisher | ND2000C | |
NaOH | Fisher | SS256 | ! CAUTION NaOH causes burns. |
NON-Fat Powdered Milk | Sangon Biotech | A600669 | |
PEG-8000 | Fisher | BP233 | |
Protein A-HRP conjugate | Invitrogen | 101123 | |
QIAprep Spin M13 Kit | Qiagen | 22704 | |
QIAquick Gel Extraction Kit | Qiagen | 28706 | |
QIAquick PCR Purification Kit | Qiagen | 28104 | |
Recombinant Protein L | Fisher | 77679 | |
T4 DNA polymerase | New England Biolabs | M0203S | |
T4 polynucleotide kinase | New England Biolabs | M0201S | |
T7 DNA polymerase | New England Biolabs | M0274S | |
Tetracycline, tet | Sigma | T7660 | 50 mg/mL in water, 0. 22 μm filter-sterilize, work concentration: 10 μg/mL. |
Tryptone | Fisher | 0123-07-5 | |
Tween-20 | Sigma | P2287 | |
Ultrapure glycerol | Invitrogen | 15514-011 | |
Uridine | Sigma | U3750 | 25 mg/mL in ethanol, work concentration: 0.25 μg/mL. |
Yeast extract | VWR | DF0127-08 | |
Name | Company | Catalog Number | Yorumlar |
Strains | |||
E.coli CJ236 | New England Biolabs | E4141 | Genotype: dut– ung– thi-1 relA1 spoT1 mcrA/pCJ105(F' camr). Used for preparation of dU-ssDNA. |
E.coli SS320 | Lucigen | 60512 | Genotype: [F'proAB+lacIq lacZΔM15 Tn10 (tetr)] hsdR mcrB araD139 Δ(araABC-leu)7679 ΔlacX74 galUgalK rpsL thi. Optimized for high-efficiency electroporation and filamentous bacteriophage production. |
M13KO7 | New England Biolabs | N0315S | |
Name | Company | Catalog Number | Yorumlar |
Equipment | |||
0.2-cm gap electroporation cuvette | BTX | ||
96-well 2mL Deep-well plates | Fisher | 278743 | |
96-well Maxisorp immunoplates | Nunc | 151759 | |
Baffled flasks | Corning | ||
Benchtop centrifuge | Eppendorf | 5811000096 | |
Centrifuge bottles | Nalgene | ||
ECM-630 electroporator | BTX | ||
Magnetic stir bars | Nalgene | ||
Thermo Fisher centrifuge | Fisher | ||
High speed shaker | TAITEK | MBR-034P | |
Microplate shaker | QILINBEIER | QB-9002 | |
Liquid handler for 96 and 384 wells | RAININ | ||
Mutil-channel pipette | RAININ | E4XLS | |
Amicon concentrator | Merck | UFC803096 |