fADL-2blue

fADL-2blue - Trypsin-Sensitive Phage Vector

Technical Description

fADL™-2blue is a type 3 phage display vector with a cloning site on the N-terminal side of the full-length gene III protein. Secretion in the periplasm of the fusion protein is driven by the PelB leader peptide, in place of the wild-type gene III protein leader sequence. fADL™-2blue derives from the phage vector fd-tet (1,2) where the tetracycline resistance genes derived from Tn10 have been replaced by the the ampicillin resistance beta-lactamase Bla gene. Display is usually highly multivalent with limited polyphage production, even in the case of large polypeptides such as scFvs. Implications of multivalent display in phage libraries versus phagemid libraries is discussed here (3). This is the recommended format for peptide libraries which will benefit of the overall improved avidity. A discussion on phage versus phagemid antibody libraries can be found here (3).

fADL™-2blue differs from fADL™-1e by the use of a different selection margler (ampicillin versus kanamycin), the formation of blue pale colonies in the presence of X-gal and IPTG, and a trypsinization site in the linker allowing elution of the bound phage by tryspin (4).

Figue 1. Display of HyHEL-10 scFv by fADL-1. Western blot analysis of phage particles prepared with fADL-1 in the presence of a plasmid supplying gene III protein (Lane A). The same blot with phage particles prepared with fADL-1 containing the HyHEL-10 scFv insert shows the scFv fused to almost all gene III proteins (Lane B). Loading is 3 x 10¹⁰ virions per lane; image was generated by ECL using a gene III protein antibody. Implications of multivalent display in phage libraries versus phagemid libraries is discussed here (3).

Applications

• Multivalent Phage display at the N-terminal side of gene III protein of the filamentous bacteriophage.

For research use only; not intended for any animal or human therapeutic or diagnostic use.

Specifications

General characteristics:

Plasmid Size: 7861
Leader Peptide: PelB
Cloning Sites: double-SfiI, BglI
Fusion Protein: full length gene III protein
Selection: kanamycin
Replication: oriF1 (60 copies/cells)

Physical characteristics:

Concentration: 0.5 µg/ µl.
Product Size: 10 µg.
Buffer: DNA Conservation Buffer (Tris/HCL 5 mM, EDTA 0.1 mM, pH 8.5, sterile).
Storage Temperature: -20°C.

Quality Control & Certification of Analysis

Product size

Digestion by ClaI,PacI and BglI generates four visible fragments of 3.3 kb, 2.0 kb, 1.6 kb and 0.9 kb.

Product sequence

Sequence of the entire vector is analyzed.

Certification

Product meets all specifications.

References

1. Zacher  3rd, A. N., Stock, C. A., Golden  2nd, J. W., & Smith, G. P. (1980). A new filamentous phage cloning vector: fd-tet. Gene, 9(1-2), 127–140.

2. Scott J. K. & Smith G. P. (1990). Searching for peptide ligands with an epitope library. Science, 249(4967):386-90.

3. O’Connell, D., Becerril, B., Roy-Burman, A., Daws, M., & Marks, J. D. (2002). Phage versus phagemid libraries for generation of human monoclonal antibodies. J Mol Biol, 321(1), 49–56.

4. Thomas WD, Smith GP. The case for trypsin release of affinity-selected phages (2010). Biotechniques, 49(3):651-4.