M13 Bacteriophage Coat Proteins Engineering

Creative Biolabs provides a method for increasing the level of protein fusions displayed on the surfaces of M13 bacteriophage particles.

Once proteins are surface displayed, methods like in vitro binding to an immobilized target molecule can be used to select proteins with high affinities. The sequences of the selected proteins can be readily deduced by sequencing the encapsulated DNA. Low copies of proteins have been displayed on M13 phage through fusion to either the P8 major coat protein or the P3 minor coat protein. Theoretically, the protein display levels should be much affected by the choice of the phagemid coat protein, since P8 is present in 2,700 copies per phage, whereas P3 5 copies per phage. However, in practice protein display levels on P8 are strongly dependent on protein sequence and length: longer polypeptides are associated with decreased display levels.

To increase the protein display level of M13 bacteriophage particles, our scientists have introduced seven-sites mutations into the N-terminal half of the P8 coat protein. This technique can work well with various proteins, and has been reported to improve the display levels of streptavidin and human growth hormone (hGH) for almost 100-fold. This dramatic improvement is because of a better accommodation of the fusion protein in the phage coat. This method allows weaker protein binders to be selected initially from both naive and small peptides libraries, and also extend phage display to the investigation of proteins with potentially novel and modified functions, which have been previously proven intractable by phage display techniques.

Fig. 1 Structure of a filamentous M13 bacteriophage. (Fukunaga and Taki, 2012)

RGD peptides have been displayed by M13 bacteriophage, and used for extracelluar matrix synthesis together with poly-lactic-co-glycolic acid nanofibers by electrospinning. The physicochemical properties and the cellular behaviors of the matrices were evaluated. The cellular behaviors and differentiation of C2C12 myoblasts have been improved on the matrices. An electrode has been designed with glucose oxidase covalently attached to gold nanoparticles assembled onto a genetically engineered M13 bacteriophage. The electrode exhibits direct electron transfer (DET) with a higher peak current per unit area of 1.2 mA/cm², and a final enzyme surface coverage of about 4.74 × 10^-8 mol/cm². That is a significant improvement over most current glucose oxidase (GOx) DET attachment methods. The M13 bacteriophage was also engineered as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein, based on the interaction between the specific phenol group of tyrosine and Fe³⁺ / Fe²⁺. We can design more exciting applications using M13 bacteriophage protein display, and bring the best out of your protein engineering projects.

Reference

Fukunaga, K. and Taki, M. (2012) 'Practical tips for construction of custom peptide libraries and affinity selection by using commercially available phage display cloning systems', Journal of nucleic acids, 2012.

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