COMPOSITE PARTS
Single Chain Variable Fragment (scFv(P5)) with CPP - Part:BBa_K3090002
scFv(P5) is a 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) scaffold is 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).
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Characterization
Vector containing insert (scFv(P5)) and pET28b (+) vector was cut by restriction enzymes (NcoI and SalI)
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After ligation, the DNA was transformed to BL21(DE3) for expression
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Expressed protein with N-terminal His6-tag was purified using Ni-NTA affinity column. CPP-scFv(P5) with molecular weight of 29 KDa was visible on the SDS-PAGE.
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The identity of CPP-scFv(P5) with N-terminal His6-tag was confirmed using anti-His6 antibody as a primary antibody for Western blotting. TEV protease with N-terminal His6-tag was used as a positive control.
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The binding between CPP-ScFv(P5)and its antigen, lysozyme was tested using a size-exclusion chromatography in reducing condition (50 mM DTT) to mimic the reducing environment of cytosol.
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The cell-penetration ability of CPP-ScFv(P5) was tested using two kinds of cell lines (HEK293T and Hela). DAPI fluorescent dye stains cell nucleus and CPP was detected by anti-his antibody and fluorescent secondary antibody.
