BASIC PARTS

Cell-penetrating Peptide - Part:BBa_K3090000

Nuclear localization signal (NLS) enriched in positively charged residues that can function as a Cell-penetrating peptide (CPP), similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT).

Characterization

This part(BBa_K3090000) was fused to the N-terminus of scFv(P5) (part number BBa_K3090001) to design a cell-penetrating antibody fragment (part BBa_K3090002). The sequence of the designed protein is as follows.

MTYTRRRFRR RRHRPRS QVQLQESGGD LVQPGGSLKL SCAVSGFSLT GYGVNWVRQT PDKRLEWVAM IWGDGNTDYN SSVKGRFTIS KDNAKSTVYL QMSSLKSEDT AMYYCARERD YRLDYWGQGT TVTVSS GGGGSGGGGSGGGGS DIELTQSPAS LAVSLGQRAT ISCRASGNIH NYLAWYQQKP GQPPKLLIYY TTTLADGIPA RFSGSGSGTD YTLTINPVEA DDVATYYCQH FWSTPRTFGG GTKLEIKR

Reference

1: Yu W, Zhan Y, Xue B, Dong Y, Wang Y, Jiang P, Wang A, Sun Y, Yang Y. Highly efficient cellular uptake of a cell-penetrating peptide (CPP) derived from the capsid protein of porcine circovirus type 2. J Biol Chem. 2018 Sep 28;293(39):15221-15232.

Single Chain Variable Fragment (scFv(P5)) - Part:BBa_K3090001

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.

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)".

Characterization

The DNA for scFv(P5) with N-terminal His6-tag was designed with codon optimization for E.coli expression.

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, 469–472.

2. Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, Benvenuto E. Engineering stable cytoplasmic intrabodies with designed specificity. J Mol Biol. 2003 Jul 4;330(2):323-32.

3. Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I., Tello, D., Dall’Acqua, W. et al. (1994). Bound water molecules and conformational stabilization help mediate an antigen–antibody association. Proc. Natl Acad. Sci. USA, 91, 1089–1093