Phage display technology has made significant progress in the field of biotechnology due to its unique advantages. This guide provides detailed answers to common questions in the technical implementation process, from library construction to antibody screening. By optimizing helper phage selection, improving display and transformation efficiency, designing high-quality primers, and employing appropriate antigen presentation and screening strategies, researchers can significantly improve library quality and screening success rates. Simultaneously, methods and measures to avoid screening for non-specific antibodies further ensure the reliability and practicality of the screening results.
A. In phage display technology, helper phages containing pⅢ phage display vectors are typically used to infect *E. coli* to prepare phage particles. The fusion protein expression in these particles exhibits a Poisson distribution; approximately 90% of the particles display only wild-type pⅢ protein, less than 10% display a single copy of the fusion protein, and displaying multiple copies is extremely rare. Therefore, most displayed fusion proteins exist in monovalent form.
Compared to multivalent display, monovalent display can remove weakly binding antibodies during the screening process. Multivalent display, due to the avidity effect, enriches weakly binding clones as well.
M13KO7, R408, and VCSM13 are helper phages containing full-length gIII, and are the most standard and commonly used type of helper phage. M13KO7 is a mutant of M13 phage, carrying a plasmid origin of replication, a kanamycin resistance gene, and the G6125T mutant gene II. VCSM13 is a mutant of M13KO7. R408 is a mutant of f1 phage, carrying an IRI mutation and lacking the antibiotic resistance marker gene. It deletes a 24bp segment from its IG, making its packaging of single-stranded DNA with a complete signal superior to its own single-stranded DNA. Overinfection using these helper phages containing full-length gIII produces helper phages and recombinant phage particles with high titers and strong target protein specificity. However, because these helper phage genomes lack packaging signals, their display level is also low, which may lead to failure in selecting specific positive clones.
M13MDD3.2, R408d3, and VCSM13d3 were constructed by deleting gIII (1579-2851) from helper phages M13KO7, R408, and VCSM13, respectively, and then using helper plasmids carrying gIII to provide the pIII protein. This ensures that during phage assembly, the sole source of pIII is the fusion protein encoded by the phage particle. Since filamentous phages have 3-5 copies of the pIII protein, this improves phage display to some extent, but the average affinity of the displayed protein is reduced. Furthermore, these helper plasmids can cause contamination to some extent, severely limiting the use of gIII-deleted helper phages.
Hyperphage was recently developed by Rondot S et al., who deleted the open reading frame (ORF) of gIII from the M13KO7 genome, retaining only the promoter and signal peptide, and adding a short peptide after the signal peptide. They constructed an *E. coli* cell packaging line (DH5α/pIII) to provide the pIII protein. This avoided the polarity effects and contamination associated with complete gIII deletion, increasing phage yield and significantly improving Hyperphage titer. CT helper phages delete the infectious N1 and N2 regions of gIII from the VCSM13 genome, retaining the CT region, and use the helper plasmid pUC19-gIII to provide the pIII protein. Due to the lack of the infection-responsible N1 and N2 regions, recombinant phages rescued by CT helper phages must embed a fusion protein to be infectious.
Helper phages Ex-phage, Phaberge, Ex12, and VCSM13N1 all introduce an amber stop codon into gIII to improve packaging efficiency.
A. Constructing high-quality display libraries requires consideration of the following five aspects:
PBMCs are crucial to the library; their total number determines the size and diversity of the library.
Use kits to extract RNA, ensuring high purity.
Antibody diversity refers to whether the primers can effectively cover the germline distribution of the species. Attention should be paid to the quality of primer design and synthesis.
Regarding primer design:
a. Primers can be designed based on germline sequences.
b. Germline expression abundance needs to be considered.
c. Degenerate primers should be avoided as they are prone to bias.
Regarding primer quality: We have found that some large domestic manufacturers, in pursuit of convenience and profit, often only desalt primers when required for PAGE or HPLC purification. Always test the primers after receiving them.
The total number of transformants determines the library size, generally requiring a library size 10 to 100 times the theoretical diversity. It's important to note that the total number of transformants does not equal the library diversity.
Background clones refer to clones without inserted fragments or clones with incorrect reading frames. These clones grow much faster than clones with correctly inserted fragments, leading to a decrease in library quality. Therefore, it is necessary to improve the insertion rate of the library and the quality of primers.
A. As mentioned earlier, the display efficiency using M13KO7 is generally low, with only about 10% of phage particles displaying antibodies, and 90% not displaying them at all. Methods to improve display efficiency include adding inducers such as IPGT to induce expression; or replacing them with helper phages with higher display efficiency, such as hyperphage and Ex-phage.
A. Selecting highly efficient electrocompetent cells can effectively improve the transformation efficiency of *E. coli*, thereby increasing the library capacity of the phage display library. Commonly used competent cells in phage display library construction include TG1, XL1-Blue, and SS320. When conducting transformation experiments, to obtain high transformation efficiency, the purity of plasmids and ligation products, the electroporation conditions, the amount of plasmids and ligation products added, and the standardization of the operation are also crucial.
A. Primer length should be 20-30 bases to ensure binding to the target sequence while minimizing interference between primers. The primer Tm value should be slightly higher than the melting temperature of the target sequence to ensure smooth binding during PCR. The GC content of the primers should be close to that of the target sequence to improve binding efficiency. Avoid primer self-complementation to prevent affecting PCR amplification. Degenerate primers should be avoided whenever possible.
A. Improper preservation methods can reduce the titer of phages. In principle, phage libraries can be preserved for a relatively long time by storing them in 50% glycerol at low temperatures (below -70℃).
A. Antigen presentation methods include direct coating, indirect coating, and screening using intact cells. Direct coating is suitable for antigens with large molecular weights and stable conformations. Indirect coating can be used for target molecules where direct coating is less effective, the antigen molecular weight is small, and the epitope is difficult to fully expose. In such cases, the biotin-streptavidin system or other tag molecules can be used to immobilize the antigen, improving the presentation efficiency. When the conformation of recombinant expressed antigens is inconsistent with that of antigens on the cell membrane, resulting in the inability to obtain antibodies binding to cell membrane surface antigens using recombinant proteins, we can use HEK or CHO cells to construct transiently or stably overexpressing cell lines, or high-expressing tumor cell lines, to present antigens and use the cells for direct screening.
A. Common screening methods include positive screening, negative screening, competitive screening, and cross-selection.
Positive screening is the most basic method, directly screening for phage antibodies that bind to the target protein.
Negative screening is a targeted method to remove non-specific antibodies. During phage antibody screening, interference from the target antigen's tag and materials, or antigens structurally similar to the target antigen, can easily lead to the selection of non-specific antibodies. In such cases, negative screening can be used to improve screening efficiency.
Competitive screening involves simultaneously binding antibodies to both the target and non-target antigens. This can screen for antibodies with higher affinity for the target antigen while eliminating antibodies bound to non-target antigens.
Cross-selection aims to reduce antibody enrichment bias. It is mainly used for antigens from different species. Cross-enrichment screening using the same antigen from different species can increase the likelihood of obtaining cross-antibodies.
A. During phage screening, many factors can lead to the selection of non-specific antibodies. We have different solutions for different situations. For example, if non-specific antibodies bind to the antigen tag are selected, we can choose to screen with a different antigen tag or perform negative screening to reduce the proportion of non-specific antibodies. If antibodies bind to the blocking solution are selected, we can choose to change to a suitable blocking solution or use different blocking solutions interchangeably to avoid this situation. If antibodies that recognize the background cell line (CHO or HEK293) are selected, we can change to a different background cell line or perform negative screening.
A. Differences in target molecule concentration, binding time, and washing intensity can all lead to variations in screening results. Antigen-antibody binding is a dynamic process. High-affinity antibodies can still bind strongly to antigens even under greater selection pressure. Therefore, we can increase the selection pressure, such as by reducing the target molecule concentration, shortening the binding time, and increasing the washing intensity, to screen for antibodies with higher affinity. Conversely, to obtain antibodies with better diversity, we need to reduce the selection pressure, especially during the first round of screening, to capture as many positive clones as possible from the constructed library. This is to maintain library diversity and ensure that the elution product in the first round is between 104-10⁷, neither exceeding nor falling below this order of magnitude.
A. After screening, the phage antibodies are packaged with helper phages and fused to the pⅢ protein for expression. We can then use enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS) for detection.
Phage ELISA is the most commonly used detection method. The antigen is coated onto an ELISA plate, blocked, and then phage supernatant is added, followed by horseradish peroxidase-labeled anti-M13 secondary antibody. Finally, color development is performed. Generally, a colorimetric value above 1.0 at OD450 is considered a positive clone.
Flow cytometry is used to detect the binding of phage antibodies to cells. After the phage supernatant binds to cells, fluorescently labeled anti-M13 secondary antibody is added. The fluorescence signal is used to determine whether the clone is positive.
Because M13 phages are filamentous and very long, they easily adhere together, leading to nonspecificity. For both natural and synthetic libraries, avoid using the phage-ELSIA method for cloning verification. Instead, utilize the 20% leakage expression of the TG1 host bacteria and then use tags on the phage particles for detection.
