Difference between revisions of "Part:BBa K3090002"

Revision as of 18:29, 23 October 2020

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

Characterization

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

After ligation, the DNA was transformed to BL21(DE3) for expression

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.

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.

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.

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.

MIT_MAHE 2020

Summary

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.

References

1. Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A., & Galeffi, P. (1993). Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature, 366(6454), 469–472. https://doi.org/10.1038/366469a0

2. Donini, M., Morea, V., Desiderio, A., Pashkoulov, D., Villani, M. E., Tramontano, A., & Benvenuto, E. (2003). Engineering stable cytoplasmic intrabodies with designed specificity. Journal of molecular biology, 330(2), 323–332. https://doi.org/10.1016/s0022-2836(03)00530-8

3. Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I., Tello, D., Dall'Acqua, W., Souchon, H., Schwarz, F. P., Mariuzza, R. A., & Poljak, R. J. (1994). Bound water molecules and conformational stabilization help mediate an antigen-antibody association. Proceedings of the National Academy of Sciences of the United States of America, 91(3), 1089–1093. https://doi.org/10.1073/pnas.91.3.1089