M13 Phage: A Versatile Building Block for Highly Specific Analytical Platform

It is well known that M13 phage has a wide range of applications in fields such as antibody discovery and vaccine development. With the deepening of research, scientists have found more and more surprises in the M13 phage. It has not only been used as M13 phage library in biological research but has now become a multifunctional construction template for a variety of applications, including sensing, imaging, therapeutics, and energy harvesting. Based on the various functions of M13 phage, Creative Biolabs provides comprehensive services to support M13 phage research.

M13 Phage

The M13 phage is a non-lytic filamentous phage with a diameter of about 6 nm and a length of about 880 nm. Its well-defined genome is packaged in five phage proteins (pIII, pVI, pVII, pVIII, and pIX), which, at the same time, determine the length of the M13 phage. Of these five phage proteins, the minor phage proteins pIII and pVI are capped at one end, while pVII and pIX are capped at the other end. There are 3–5 copies of each minor phage protein. Each pVIII consists of three structural domains—a positively charged domain (40–50 AA) that interacts electrostatically with phage genomic DNA, an intermediate hydrophobic domain (21–39 AA), and an N-terminal domain (1–20 AA). Through the electrostatic interaction between phage genomic DNA and the positively charged structural domain of pVIII protein, a total of 2,700 pVIII protein helices were entangled in phage DNA, resulting in a negative charge on the M13 surface.

Fig 1. An illustration of the structure of M13 phage and the amino acid sequence of pVIII protein. B Schematic showing the infection and production process of natural M13 phage. C Illustration of the mechanism of phage vector-based display system. D Illustration of the mechanism of phagemid-based display system. (Wang, R., et al., 2023)

A unique advantage of phage over other man-made biomaterials is its ability to display proteins/peptides on desired coat proteins through genetic modification. An important method to allow M13 phages to display exogenous proteins and peptides is the phage display technology, known as biopanning. This is an evolutionary method for selecting peptide ligand diversity through high throughput and has become an indispensable tool for exploring specific biochemical molecular interactions. There are two main approaches to genetically modifying M13 phage, i.e., surface display via phage vectors or phagemid.

Engineered M13 Phage

Using genetic engineering and chemical modification, different types of M13 phage coat proteins can be manipulated to have desired functions, endowing the M13 phage to serve as a multifunctional analysis platform with different functional regions that perform their functions without interfering with each other. The use of engineered M13 phage in analytical applications gains particular benefits due to its unique features, such as filamentous morphology and flexibility.

For analyte isolation and separation, M13 phage, with its unique characteristics of filamentous morphology and orthogonal reactivity, can be used as an ideal alternative for constructing immunosorbents for analyte separation. The target binding ability can be obtained by chemically coupling of the binding ligands (antibodies, aptamers, etc.) on the phage surface. In addition, by genetically modifying the M13 phage to display 6His on the PIII protein, terminal anchoring of M13 is achieved through 6His-NTA interactions, which provides a strong and reversible assembly. The terminal approach also facilitates the construction of bio-inspired structures that mimic cellular thread-like structures and allow high binding to functional molecules. Biotinylation of M13 phage by displaying a 14-mer biotin receptor peptide on the PIII protein with the help of the biotin ligase BirA was used for M13 terminal anchoring on streptavidin-functionalized microbeads.

In terms of immunoassays, M13 phages can function as capture antibodies and signaling antibodies, among others. M13-based capture antibodies utilize the multivalency of the PVIII display system to enhance analyte capture, while M13-based signaling antibodies benefit from the 5 : 2700 molecular ratio between PIII and PVIII for signal amplification.

Reference

Wang, R., et al., M13 phage: A versatile building block for a highly specific analysis platform. Anal Bioanal Chem, 2023. 415(18): p. 3927-3944.

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