Difference between revisions of "Part:BBa K3398003"

Latest revision as of 11:42, 26 October 2020

mamC_ZZ fusion protein

This part is encodes a fusion protein of mamC and ZZ. And for separation from other proteins at the purification step, we introduced a GST tag at the N-terminus and a thrombin site between the fusion protein and tag for cleavage after purification, which has been submitted as BBa_K3398004. To maximize the expression of a functional protein, we employed codon optimization to maximize the expression of a functional protein in Escherichia coli. Not only in E.coli, these parts can also be expressed by magnetotactic bacterium such as MSR-1 to produce magnetosomes modified with ZZ protein on the surface as a self-assembly platform for polymer varied antibodies.

Usage and Biology

MamC protein is a membrane protein which covers the most area of the magnetosome surface formed by magnetotactic bacteria MSR-1, and ZZ is the Fc-binding domain of staphylococcal protein A (SPA), an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus. This fusion protein can be expressed and anchored onto the surface of magnetosomes by transferring the recombinant plasimd into MSR-1.The resulting recombinant magnetosomes will be capable of self-assembly with the Fc region of mammalian antibodies and will ve therefore useful for functionalization of magnetosomes such as being developed to a antibody vector or serving as specifically tumor-targeting contrast agents for MRI.

Characterization

Plasmid construction

We designed the fusion protein sequence ordered gene synthesis service from GenScript Biotech Corporation and obtained the recombinant pGEX-2TK plasmid with a GST 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 mamC-ZZ vector.

Figure 2. The result of PCR for mamC-ZZ.

Expression of mamC_ZZ

After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3). The plasmid was transferred into E.coli BL21(DE3) and massively expressed after the addition of IPTG. 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 3 and Figure 4 below, and it shows that the best concentration of IPTG for induing is 2mM and the optimum inducing time is 4 hour.

Figure 3. Western-blot results of GST mamC-ZZ under different inducing IPTG concentration.

Figure 4. Western-blot results of GST mamC-ZZ under different inducing time.

Purification of mamC_ZZ protein

Immunoprecipitation (IP) was performed to obtain purified GST-tagged protein and investigate the optimum condition of purification. IP has been described in detail in the experiment section in our wikis. The target protein appeared at 52kDa. A group of gradient glutathione resin, which added as single,double and triple volume of GST mamC-ZZ solution, was tested. The results of IP are shown in Figure 5 below the efficiency of purification did not demonstrate a significant difference among different glutathione resin volume which added in. In following work, we added equal glutathione resin as GST mamC-ZZ solution to purify the recombinant protein.

Figure 5. Coomassie staining results of GST mamC-ZZ under different purification conditions.

Co-immunoprecipitation with scFv_Fc

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 6 and Figure 7 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 6. Western-blot results of mamC-ZZ and scFv which introduce different primary antibody.

Figure 7. Western-blot results of input control block.

Sequence and Features