Q1: What is phage display technology?

A1: Phage display technology involves cloning the coding gene or target gene fragment of a peptide or protein into an appropriate location within the structural gene of a phage coat protein. Under the condition that the reading frame is correct and the normal function of other coat proteins is not affected, the foreign peptide or protein is expressed as a fusion with the coat protein. The fusion protein is then displayed on the surface of the phage during the reassembly of progeny phages. The displayed peptide or protein retains its relatively independent spatial structure and biological activity, facilitating the recognition and binding of target molecules.After incubating the peptide library with the target protein molecule immobilized on a solid phase for a certain period, unbound free phages are washed away. The phages bound to the target molecules are then eluted using competitive receptors or acid treatment. The eluted phages infect host cells, undergo amplification, and proceed to the next round of elution. After 3 to 5 rounds of "adsorption-elution-amplification," the phages specifically binding to the target molecule become highly enriched. The resulting phage preparation can be used for further enrichment of target phages with desired binding properties.

Q2: What are the advantages of phage display compared to other antibody development technologies?

A2: Phage display technology offers significant advantages over hybridoma technology. The hybridoma method is only applicable to mice, rats, hamsters, and guinea pigs. In contrast, phage display can be used to generate high-affinity monoclonal antibodies in all commonly used antibody-producing species, including but not limited to humans, mice, rats, rabbits, chickens, camels, llamas, alpacas, cattle, dogs, sheep, monkeys, and sharks.Hybridoma-based monoclonal antibody development typically yields only a small number of antibodies against a specific immunogen at a time, whereas phage display technology can present the entire antibody repertoire of an immunized animal, with nearly 10% of the antibodies being immunogen-specific. The chances of discovering antibodies with desired properties are much higher using phage display.Additionally, with hybridoma technology, it is difficult to incorporate enrichment steps that selectively isolate antibodies with desired functions. In most cases, all hybridoma clones are first generated and then validated one by one. In contrast, phage display allows for various enrichment strategies: antibodies with desired properties can be enriched, while those lacking the desired functions can be excluded from further validation. For example, antibody library screening can use target capture to isolate strong binders while employing controls to block or deplete cross-reactive binders.In summary, this immune antibody library approach enables the collection of nearly all antibodies within an animal and the isolation of the strongest binders from the collection.

Q3: What are the differences among the M13, T7, and T4 phage systems?

A3: The M13 filamentous phage has been the most popular choice and is widely used in various types of research. The viral coat consists of five distinct capsid proteins, including a major capsid protein pVIII (2,700 copies) and four minor capsid proteins (pIII and pVI at one end, and pVII and pIX at the other). Unlike T4 and T7, M13 is a lysogenic phage that assembles in the periplasm and is secreted from the bacterial membrane without lysing the host. The multiple capsid proteins on M13 phage provide a comprehensive selection for displaying diverse peptides and proteins with unique properties. All five capsid proteins have been successfully used for foreign domain display with distinct vectors. Among them, pIII and pVIII are the primary choices for M13 phage display.

The T4 phage differs from M13 in many aspects. T4 is larger in size, featuring a tail structure and a double-stranded DNA (dsDNA) genome encoding 50 different proteins. The larger genomic DNA allows for the insertion of bigger foreign proteins. Two distinct domains can be displayed on HOC and SOC, both of which are non-essential capsid proteins. Both N- and C-terminal insertions are possible. Since there is no membrane secretion process, using T4 phage avoids host toxicity.

Compared to filamentous phages and λ phage, the T7 phage has a shorter life cycle. Progeny phage assembly occurs in the bacterial cytoplasm, and release is achieved through cell membrane lysis, eliminating size restrictions and host toxicity caused by secretion processes. Additionally, T7 phage is highly stable under extreme conditions where other phages cannot survive.

Q4: Can the M13 phage display system be used to construct a cDNA library?

A4: M13 is generally not suitable for cDNA expression because the M13 phage display system requires in-frame expression between the leader sequence (necessary for secretion) and the N-terminus of the capsid protein pIII or pVIII. To properly fuse the target protein with the capsid protein, the insert must be in the correct reading frame at both ends and must not contain in-frame stop codons. These constraints result in an extremely low number of viable clones in an M13 cDNA library.

Q5: What type of culture medium can be used for phage propagation?

A5: TSB/TSA can be used to culture most phages. However, the optimal medium may vary depending on the specific phage display system.

Q6: Which strains of E. coli can bacteriophage M13 infect? Is there a chance it could contaminate all the cell lines in our lab?

A6: M13 phage is a filamentous bacteriophage that uses the tip of the bacterial F-pilus as a receptor to facilitate the infection process. Therefore, it is specific only to E. coli strains carrying the F-plasmid (F+). For the strains in your lab, we recommend checking whether they contain the F-plasmid. If they do not, M13 phage cannot infect them.

Q7: Does the E. coli TG1 host strain contain antibiotic resistance?

A7: The E. coli TG1 strain does not possess antibiotic resistance, while the phage-infected TG1 strain can be selected using 2YT-AK medium (2YT supplemented with 100 μg/mL ampicillin and 50 μg/mL kanamycin).

Q8: What is the difference between PFU and CFU?

A8: PFU (Plaque-Forming Unit) measures the number of individual infectious particles and is commonly used to quantify bacteriophages. CFU (Colony-Forming Unit) measures viable cells, where a colony represents a cluster of cells derived from a single progenitor cell, and is typically used for bacterial counting (e.g., E. coli).

Q9: How to evaluate the quality of a constructed phage display library?

A9: The affinity of antibodies selected from an immune library is proportional to the library size—the larger the library, the better the antibody affinity. Typically, the final library size should reach 10⁸, which is sufficiently large to isolate high-affinity antibodies. Additionally, a high-quality library should exhibit high diversity. Quality control (QC) results of the final library should show no common sequences among randomly picked clones, indicating a very high level of diversity.

KMD Tech specializes in the biotechnology sector, offering high-efficiency phage display technology services backed by a professional team and cutting-edge expertise. Our services encompass the entire workflow, from the design and synthesis of target proteins to the construction and screening of phage libraries, as well as the identification and optimization of high-affinity antibodies.Our technology enables the precise identification and screening of antibodies that exhibit strong binding to specific antigens from vast phage libraries, significantly enhancing the efficiency and success rate of antibody discovery. Additionally, KMD Tech provides follow-up services, including antibody humanization, expression, and purification, delivering a one-stop solution from lab research to industrial applications in fields such as drug development and disease diagnostics.Committed to advancing phage display technology, we empower researchers and pharmaceutical companies to accelerate novel drug development and drive progress in biomedical research.