Difference between revisions of "Part:BBa K3398005"

Latest revision as of 12:30, 26 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

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.

Figure 1. Map of scFv-Fc vector.

Figure 2. The result of PCR for scFv-Fc.

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.

Figure 3. Coomassie staining results of scFv-Fc to investigate the solubility.

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.

Figure 4. Western blotting results of scFv-Fc under different inducing concentration of IPTG.

Figure 5. Western blotting results of scFv-Fc under different induction time.

Purification of scFv-Fc

We perform the immunoprecipitation (IP) to obtain purified FLAG-tagged protein. IP has been described in detail in the experiment section in our wikis.The results of IP are shown in Figure 6 below. The target protein appeared at 54kDa. Nevertheless, there were a lot of proteins in the high molecular weight part, because scFv would form polymer, which was immobilized as well. At the same time, there was protein in small the molecular weight part, which may be caused by the protein truncating easily at the junction.

Figure 6. Western blotting results of immunoprecipitation of scFv-Fc.

Co-immunoprecipitation with mamC-ZZ

To demonstrate that mamC_ZZ and scFv_Fc truly bind together and test the binding specificity. We carried out Co-IP to see if there is interactions between these to fusion proteins, which support our future work that constructing functionalized magnetosomes. The supernatant of cell lysate of mamC-ZZ or purified mamC-ZZ was incubated with purified FLAG-tagged scFv-Fc overnight at 4 °C. Afterwards, the mixture was incubated with anti-FLAG resin (GenScript, Nanjing, CN) for 1 h. The FLAG-tagged proteins scFv-Fc and the interacted mamC-ZZ from the lysate were then immobilized on the resin, whereas the unbound proteins were washed away with TBS. Subsequently, the protein–protein complex was eluted with acid elution buffer. The elution was then digested by thrombin to cut off GST region. The product was then analyzed by SDS-PAGE and western blotting. For negative control, only purified scFv-Fc was used as input. The results of Co-IP are shown in Figure 7 and Figure 8 below,the mamC-ZZ could bind secondary antibody by Fc region directly and the band would appear at 26 kDa approximately. The result shows that lane 2 indicated that scFv-Fc was immobilized on the resin, meanwhile, the ZZ region specifically interacted with the Fc region and also showed a band. Furthermore, lane 3 suggested a more inspiring result, which meant scFv-Fc could bind mamC-ZZ in a complicated environment. These results implied us an easier way to purified and enriched mamC-ZZ from cell lysate directly.

Figure 7. Western-blot results of mamC-ZZ and scFv which introduce different primary antibody.

Figure 8. Western-blot results of input control block.

Sequence and Features