Superphage is a derivative of M13KO7. In bacteria, the wildtype pIII level from M13KO7 is much higher than that of antibody-pIII fusion protein from phagemid. Moreover, phage assembly has a preference for the wild-type pIII even if the wild-type pIII and fusion pIII are at the same level. As a result, the antibody display level is about 1% in an M13KO7-packaged phage library, indicating that 99% of the library is "bald" and doesn't display any antibodies [1-2]. The low display level results in low success rate of isolating specific antibodies [3].

Figure 1. Superphage dramatically decreases wildtype pIII expression and increases scFv display on the phage. (A) TG1 cells were transformed with phagemid pFSA which expressed scFv-pIII fusion protein The cells grow in 2YT media containing 2% glucose and 100ug/ml carbenicillin until OD600 ~0.5. Divide the cell culture into 3 portions, and add VCSM13, M13KO7, and Superphage at a multiplicity of infection (MOI) of 10:1 (phage-to-cells ratio) to the culture respectively. Shake at 60rpm for 15min and then at 250rpm for 45min. Change media to 2YT containing 500ug/ml IPTG and 100ug/ml carbenicillin and 50ug/ml kanamycin. Shake at 250rpm for 5 hours and collect bacteria for western blot with anti-pIII mAb to compare the level of wild-type and fusion pIII. The result showed that Superphage dramatically decreased wild-type pIII expression compared to M13KO7. (B) After overnight culture, phages were purified from bacterial supernatant, and analyzed by western blot with anti-pIII mAb to estimate scFv display level. In Superphage-packaged phages, the level of scFv-pIII fusion protein is about one-third of wildtype pIII. Usually, there are 3-5 copies of pIII on each mature phage, indicating that one copy of scFv-pIII fusion protein is displayed on the surface of each phage averagely. In M13KO7/VCSM13-packaged phages, the scFv display level is much lower. 

Mechanism: Superphage dramatically suppresses the expression of wildtype pIII (Figure 1A). The level of wildtype pIII is so low that it is not enough for phage assembly and more antibody-pIII fusion protein is forced to participate in the process. As a result, the antibody display level was increased about 100-fold compared to M13K07-packaged phages. Superphage-packaged phages have the following features: (1) The antibody display level is ~100% (Figure 1B); (2) It is  still monovalent display, good for selecting high-affinity antibodies; (3) Each phage has multiple wild-type pIII  so that phage infectivity is not compromised; (4) The phage titer is  high; about 10¹¹ cfu/ml; (5) In Superphage-packaged phage library, the ratio of phagemid particles to phage particles is 500000:1. In contrast, the ratio is about 10:1 in the M13KO7-packaged phage library; (6) Superphage is compatible with all phagemid and bacterial strains, unlike Phaberge, EX-phage, and Ex12 which works only in the amber non-suppressor strains.

The coupling of phenotype and genotype is not always true [4]. During a library assembly, the displayed protein may be either wild type (derived from the helper phage) or antibody-pIII fusion protein (derived from the phagemid), and the packaged genome may be either phage or phagemid. Therefore, there are four kinds of virus particles in an antibody phage library. (i) phagemid genome and antibody-pIII fusion protein (the desired result). (ii) phagemid genome and wild type pIII (unable to bind the target and will be washed away during the panning). (iii) phage genome and antibody-pIII fusion protein (possible to bind a target but cannot be amplified). (iv) phage genome and wild type pIII (the helper phage itself). In theory, the mutated packaging signal in helper phage should significantly reduce the number of helper phage particles during the library assembly. However, the number of phage particles can sometimes equal, or exceed, the number of phagemid particles, which can significantly compromise subsequent selections. The ratio of phagemid particles to phage particles depends on both helper phage and phagemid vector. In pFSA/Superphage system, the ratio of phagemid particles to phage particles was 500000:1 (Table 1), and almost all the virus particles are phagemid particles. 

Table 1.  Comparison of features of helper phages. 

Table 2.  Specifications of Superphage

References

1. Kramer RA, Cox F, van der Horst M, et al. A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein. Nucleic Acids Res.  2003; 31(11): e59. 

2. Azzazy HM, Highsmith WE Jr. Phage display technology: clinical applications and recent innovations. Clin Biochem. 2002; 35(6): 425-45.

3. Baek H, Suk KH, Kim YH, et al. An improved helper phage system for efficient isolation of specific antibody molecules in phage display. Nucleic Acids Res.  2002; 30(5): e18. 

4. Chasteen L, Ayriss J, Pavlik P, et al. Eliminating helper phage from phage display. Nucleic Acids Res. 2006; 34(21): e145.