Technical Description
pADL™-22c is a phagemid vector designed for phage display on the N-terminal side of the protein III of the filamentous bacteriophage M13 or equivalent. This vector contains a PelB leader sequence for expression in the periplasm, a double-SfiI cloning site to introduce scFvs or Fab fragments, a HIS tag for purification, a HA tag for detection and an amber codon located before the full-length copy of the gene III sequence. On non-suppressive bacterial strains, free scFvs or Fab fragments are produced in the periplasm where they can be assayed for binding or purified for further testing. Expression of the fusion is under the control of a lac promoter that has been adjusted at a level comparable to the widely used phagemid pCOMB3.
Figure 1. Reactivity of HA tag. Western blot analysis of periplasmic extracts prepared from varied phagemids containing the HyHEL-10 scFv insert. Only the scFv derived from pADL-22 exhibits a strong reactivity with a HA tag antibody (Left Panel; Lane A: pADL-20, Lane B: pADL-22, Lane C: pADL-23). Binding analysis of HyHEL-10 scFv by ELISA using a HA tag antibody as a secondary antibody (Right Panel; PERI: periplasmic extract, SUP bacterial supernatant, LYZ: lyzozyme, BSA: bovine serum albumin). Note: Expression of pADL-22c is 5 to 10 times higher than the parent vector pADL-22.
Applications
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Phage display at the N-terminal side of gene III protein of the filamentous bacteriophage.
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Free scFv or Fab expression.
Specifications
General Characteristics:
Plasmid Size: 3957
Promoter: lac promoter
Leader Peptide: PelB
Cloning Site: double-SfiI/BglI, NotI-SpeI
Purification: HIS tag
Detection: HA tag
Fusion Protein: full length gene III protein and conditional Amber stop codon
Selection: ampicillin
Replication: oriF1, pMB1
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.
