Part:BBa_K3090002

single chain variable fragment (scFv(P5)) with cpp

scFv(P5) is chimeric molecule in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffoldis to maintain cytoplasmic stability and specificity. Cell penetrating peptide from Porcine circovirus type 2 was fused to the N-terminus of scFv(P5) for cell penetration.

Usage and Biology

Only a few antibodies have proved to possess naturally both high in vitro thermodynamic stability and the capacity to be functionally expressed in the cytosol milieu. Among these, the scFv(F8), deriving from a monoclonal antibody raised against the coat protein of the plant virus AMCV, has been expressed as a functional molecule in the cytoplasm of Escherichia coli, yeast,and plants. Denaturation/renaturation studies indicate that this molecule has high in vitro stability and is capable of refolding to a functional form under reducing conditions. Based on the scFv(F8) scaffold, antigen binding residues in the complementarity determining regions (CDRs) of anti-hen egg white lysozyme (HEL) D1.3 monoclonal antibody was grafted to scFv(F8) to make this part "scFv(F8)". Cell-penetrating peptide (part number BBa_K3090000) was fused to the N-terminus of scFv(P5) (part number BBa_K3090001) to create a cell-penetrating antibody fragment (part number BBa_K3090002).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 306
  

Characterization

Vector containing insert (scFv(P5)) and pET28b (+) vector was cut by restriction enzymes (NcoI SalI)