Difference between revisions of "Part:BBa K929101"

Revision as of 17:49, 1 October 2012

Anti-EGFR scFv425 with TEV site, transmembrane region an eYFP

General information

Anti-EGFR scFv425 with TEV site, transmembrane region an eYFP
BioBrick Nr.	BBa_929101
RFC standard	RFC 25
Requirement	pSB1C3
Source	BBa_K157001, BBa_K929100, BBa_K157010, BBa_E0030
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

The Biobrick BBa_K929101 is an extended version of the Biobrick BBa_K929100 and is composed of the already existing human EGFR (ErbB-1) signal peptide (BBa_K157001), the scFv anti-EGFR-domain-3, a TEV recognition site (according to life technologies AcTEV TM Protease manual), the modified transmembrane domain: transmembrane domain of B-cell receptor with glycine-serine linker (BBa_K157010) and the enhanced YFP (BBa_E0030) reporter. The composite part was derived by PCR assembly.

Signal peptide:
The human EGFR (ErbB-1) signal sequence fused to the N-terminus of a fusion protein with a transmembrane domain mediates the transport to the membrane (BBa_K157001).

Anti-EGFR scFv425:
The single chain fragment (scFV425) specifically binds to the epidermal growth factor receptor (EGFR) domain 3. It has a shortened N-terminal FLAG-tag sequence of five amino acids (DYKDE) for purification and detection.

TEV recognition site:
The TEV protease cleavage site permits the shift from surface presentation of the scFv antibody to a secretory scFv production on protein level. The TEV protease recognition site ENLYFQG is the most commonly used aa sequence for recognition by the 27kDA catalytic domain of Nuclear Inclusion a (NIa) protein encoded by the tobacco etch virus (TEV). This sequence is extended by a 3 aa linker at the N-terminal and a 2 aa linker at the C-terminal end.

Transmembrane domain of B-cell receptor with glycine-serine linker:
Helical single-span transmembrane domain of the B-Cell-Receptor with a flexible 3 aa linker at the C-terminal end (modified BBa_K157010). This part was designed for fusion to proteins or peptides that will be presented on the cells surface. A signal peptide at the N-terminal end of the fusion protein for membrane integration (e.g. part BBa_K157001) is highly required.

Reportergene: Enhanced YFP ( BBa_E0030)
Expression of the scFv fusion protein and its cellular localization can be monitored by the enhanced YFP signal with an excitation at 514 nm and an emission at 527 nm.

Characterization

Yellow fluorescence reporter and expression in CHO-cells:

Fig. 2: eYFP fluorescence of the small construct in transiently transfected CHO cells

The antibody construct was designed with a eYFP reporter to check the expression level of the construct, its cellular localization and to enable the selection of transfected cells via FACS.

The fluorescence microscopy image shows transfected CHO cells with the antibody construct containing the scFV425 (BBa_K929101). It can be proven that the construct is expressed in CHO cells and also shows a high fluorescent signal. Its accumulation in the vesicular compartment of the golgi apparatus implements a membranous localization.

Sequence and Features