pFSA is a phagemid vector designed for high-level expression and display of antibody fragment-pIII fusion protein. The Display level depends on phagemid vector as well as helper phage. pFSA significantly increased the expression level of scFv-pIII expression level (Figure 1) and display level (Figure 2). 

                                             (samples: bacterial lysates)

Figure 1. pFSA significantly increased the expression level of scFv-pIII fusion protein. TG1 cells were transformed with 4 different phagemid vectors phagemid1, phagemid2, phgemid3, and pFSA respectively. All of them expressed the same scFv-pIII fusion protein. The cells were cultured in 2YT media containing 2% glucose and 100ug/ml carbenicillin until OD600 ~0.5. The culture was divided equally and infected by M13KO7 and Superphage 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. The bacterial lysates were analyzed by western blot with anti-pIII mAb to detect the expression level of wild-type and fusion pIII. The result showed that (1) pFSA significantly increased the expression of scFv-pIII fusion protein compared to phagemid1, phagemid2, and phgemid3. (2) Superphage dramatically decreased the expression of wild-type pIII expression compared to M13KO7.  

                                               (samples: phage lysates)

Figure 2. pFSA significantly increased the display level of scFv-pIII fusion protein on the surface of M13 phage. TG1 cells were transformed with 4 different phagemid vectors phagemid1, phagemid2, phgemid3, and pFSA respectively. All of them expressed the same scFv-pIII fusion protein. The cells were cultured in 2YT media containing 2% glucose and 100ug/ml carbenicillin until OD600 ~0.5. The culture was divided equally and infected by M13KO7 and Superphage 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 overnight at 250rpm and 30℃. Phages were purified from bacterial supernatant, and analyzed by western blot with anti-pIII mAb to determine scFv display level. The results showed that (1) the display level of scFv-pIII was the highest with pFSA, and the display level with phagmide1 and phagemid2 were too low to be detected by western blot, and (2) Superphage dramatically increased the display level of scFv-pIII. 

In phagemid/helper phage system, each E. coli contains two kinds of pIII for phage assembly (e.g. wild-type pIII derived from the helper phage and antibody-pIII fusion protein derived from the phagemid), and two kinds of genomes for packaging (e.g. ampicillin-resistant ssDNA derived from phagemid and kanamycin-resistant ssDNA derived from helper phage). Therefore, there are four kinds of virus particles in an antibody phage library: (1) phagemid genome and antibody-pIII fusion protein (the desired result). (2) phagemid genome and wild type pIII (unable to bind the target and will be washed away during the panning). (3) phage genome and antibody-pIII fusion protein (possible to bind a target but cannot be amplified). (4) 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[1].  

To analyze the ratio of phagemid particles to phage particles, bacteria were infected with the virus particles. Spread the infected bacteria on carbenicillin plates to calculate the titer of phagemid particles (Table 1), and on kanamycin plates to calculate the titer of phage particles (Table 2). The ratio of phagemid particles to phage particles depends on both helper phage and phagemid vector. In the pFSA/Superphage system, the ratio of phagemid particles to phage particles was 500000:1 (Table 3), and almost all the virus particles are phagemid particles. 

Table1. Titer of phagemid particles (cfu/ml)

Table2. Titer of phage particles (cfu/ml)

Table3. The ratio of phagemid particles to phage particles

Table4. Specifications of pFSA

References

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