Difference between revisions of "Part:BBa K3398005"

Revision as of 08:26, 22 October 2020

scFv_Fc fusion protein

This part is encodes a fusion protein of single-chain variable fragment (scFv) and fragment crystallizable (Fc). And for separation from other proteins at the purification step, we introduced a FLAG tag at the C-terminus which was followed by an enterokinase cleavage site for cleavage after purification, which has been submitted as BBa_K3398006. To maximize the expression of a functional protein, we employed codon optimization to maximize the expression of a functional protein in Escherichia coli. As the redox potential of general E.coli such as BL21 is too high to form the disulfide bond in scFv, which may not express the functional fusion proteins. So we chose E.coli Shuffle with a reduced cytoplasmic environment as the chassis organism to express scFv_Fc fusion protein. These parts are ideal molecule which bind HER2 cells strongly and specifically.By adding report genes at either C- or the N-terminus, they can be used for the detection of HER2 cells.

Usage and Biology

For cloning of the fusion of single-chain variable fragment (scFv) and fragment crystallizable (Fc), the amino acid sequence of anti-HER2 scFv described by Ahmadzadeh, M. [1]and the CH2 & CH3 region of immunoglobulin heavy constant gamma 1, which is the constant region of immunoglobulin heavy chains was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1 was used as the linker between scFv and Fc.As the redox potential in the cytoplasm of general E.coli such as BL21 is too high, the disulfide bond in scFv will be reduced into thiol, which may not express the functional fusion proteins. So if you want to express this protein, we recommend using E.coli SHuffle® with a reduced cytoplasmic environment as the chassis organism to express scFv-Fc fusion protein. This part is an ideal molecule which can bind HER2 cells strongly and specifically, by adding report genes at either C- or the N-terminus, they can be used for the detection of HER2 cells.

Characterization

plasmid construction

Sequence and Features