Part:BBa_K3398005
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
We designed the fusion protein sequence ordered gene synthesis service from GenScript Biotech Corporation and obtained the recombinant pET19b plasmid with a 10× histidine tag . To confirm the sequence of the fusion protein, we designed a couple of primers to amplify the sequence by PCR and sent to Zhejiang Sunya Biotechnology Co., Ltd for sequencing.The vector map and PCR results are shown in Figure 1 and Figure 2 below.
Expression of scFv-Fc
After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the recombinant plasimid pET19b contains scFv-Fc was transformed into E.coli SHuffle and massively expressed after the addition of IPTG. To indicate the solubility of scFv-Fc, the cells were cultured in supernatants and pellets, and the cell lysate was analyzed by SDS-PAGE respectively. The Coomassie staining results of scFv-Fc are shown in Figure 3 below. To investigate the optimum concentration of IPTG and the optimum time for inducing, we carried out gradient experiments at the same time. The results of concentration and time gradient experiments are shown in Figure 4 and Figure 5 below, and it shows that the best concentration of IPTG for induing is 2mM and the optimum inducing time is 24 hour. There was a small amount of expression without IPTG induction, probably due to promoter leakage. The expression level of IPTG decreased first and then increased with the increase of IPTG concentration. This may cause by the inhibitory effect of IPTG on bacterial growth.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1074
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 741
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