Difference between revisions of "Part:BBa K929107"

Latest revision as of 18:43, 1 October 2012

Anti-GFP, IgG1 Fc with TEV site, TMD, mCherry, framed by LoxP

General information

Anti-GFP, IgG1 Fc with TEV site, TMD, mCherry, framed by LoxP
BioBrick Nr.	BBa_929107
RFC standard	RFC 25
Requirement	pSB1C3
Source	Gene synthesis by GeneArt
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

The antibody construct consists of two major building blocks represented by the actual antibody unit ( BBa_K929102) and the switchable membrane anchoring region (BBa_K929103). Both elements guarantee the eligibility and the easy handling of the construct and are optimized for expression in CHO cells. The antibody unit is represented by the human Ig kappa chain V-I region signal peptide (UniProt: P01601), the anti-GFP Nanobody (PDB: 3OGO) and the Fc region (UniProt: P01857). TEV protease recognition site, 2 LoxP sites, the B-cell receptor transmembrane domain (modified BBa_K157010) and the mCherry reporter display the switchable membrane anchoring region.
This part represents an antibody unit composed of a IgG kappa signal peptide, an anti-GFP nanobody and an IgG1 Fc domain.

Signal peptide IgG kappa:
The human signal peptide of the immunoglobulin G kappa variable chain I has 22 aa and is responsible for the translocation of the fusion protein to the membrane (UniProt: P01601).

Anti-GFP nanobody:
The green fluorescent protein (GFP)-nanobody (PDB: 3OGO) is a single-chain VHH antibody domain developed with specific binding activity against GFP and shows a Kd value of 1.4 nM. Its CDR3 loop is very short and has significantly fewer contacts with the GFP ligand compared to other nanobodies. Furthermore, the shortness of this CDR3 loop leads to the exposure of the framework 2 region, which has a major contribution to the binding with GFP.

Fc region:
A human immunoglobulin gamma-1 heavy chain constant region (IGHG1) was added to the construct and enables the direct interaction of the antibody unit with the Fc receptor and complement proteins ( UniProt: P01857).
TEV recognition site:
The TEV protease cleavage site permits the shift from surface presentation of the nanobody-Fc fusion protein to a secretory 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.

LoxP site:
The membrane anchoring region is flanked by two LoxP sites introducing a non-reversible genetically switch to our system which allows the modification from surface presentation to secretion on the DNA level. Both LoxP sites are oriented in the same direction leading to the elimination of the enclosed region by cre recombinase activity.

Transmembrane domain of B-cell receptor with glycine-serine linker: modified BBa_K157010
Helical single-span transmembrane domain of the B-Cell-Receptor with a flexible 3 aa linker at the C-terminal end. 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.

mCherry:
Expression of the switchable membrane anchoring region and its cellular localization can be monitored by the mCherry signal with an excitation at 587 nm and an emission at 610 nm.

Characterization

mCherry reporter and expression: in CHO-cells:

Fig. 1: mCherry fluorescence of advanced antibody construct showing membrane localisation on CHO cells

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

The fluorescence microscopy image shows transfected CHO cells with the antibody construct containing the anti-GFP nanobody (PDB: 3OGO). The images indicate that the construct is expressed in CHO cells and shows a high fluorescent signal sufficient for detection. With fluorescence microscopy, confocal microscopy, immunfluorescence and FACS we were able to show the expression of the transfected parts. Under certain conditions we have seen a membrane localization of the advanced construct yet we have not been able to generate cells that transport enough molecules to their surface for successful detection.

Functionality of Applied Cre/LoxP System in CHO-cells:

Fig. 2: Western Blot of purified Nanobody-Fc from supernatant of cells co-transfected with Cre recombinase and advanced construct

As an additional functional unit the antibody construct contains two LoxP sites which flank the membrane anchoring region, thus allowing the switch from surface presentation to secretion on genetically level.

By co-transfecting CHO cells with the antibody construct and the cre recombinase (pBS185) we were able to proof the functionality of the applied cre/LoxP system. The immune blot image (Fig. 2) shows purified Nanobody-Fc from supernatant obtained from co-transfected CHO cells colored with anti-human Fc coupled to streptavidin. Purification was carried out with magnetic beads. A correct band can be seen at 41kDa.

Usage and Biology

Sequence and Features