Team:ZJU-China/Experiments
Experiments
Cloning
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 (UniProtKB-P01857), was used respectively. In addition, the hinge region of immunoglobulin heavy constant gamma 1 was used as the linker between scFv and Fc, a FLAG tag was fused to the C-terminus as well. Furthermore, we employed codon optimization to maximize the expression of a functional protein in Escherichia coli. The gene was synthesized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pET19b, introduced a 10× histidine tag at the N-terminus.
Figure 1. Vector of scFv-Fc, pET19b.
For cloning of the fusion of mamC (GeneBank: AVM72836.1) and ZZ (GeneBank:M74186.1 ), the gene was synthesized and codon optimized by GenScript Biotech Corporation (Nanjing, CN). The gene was subcloned into gene expression vector pGEX-2TK, introduced a GST tag at the N-terminus.
Figure 2. Vector of mamC_ZZ, pGEX-2TK.
Expression of scFv-Fc
After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pET19b was transformed into Escherichia coli SHuffle®T7 Express Cells. The cells were grown in LB medium at 37°C until the OD600 reached 0.6, at which time protein expression was induced by adding filter sterilized isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then incubated at 30°C for an additional time to expression. Afterwards, the cells were harvested by centrifugation at 4000 rpm for 12 min. The cell pellet was resuspended in lysis buffer (50 mM Tris/HCl, pH 7.5, 500 mM NaCl, 0.1% CA630, 10% glycerol and 5 mM β-mercaptoethanol) and the cells were lysed by sonication. The cell extraction was clarified by centrifugation at 10000 rpm for 15 min.
Expression of mamC-ZZ
After the DNA sequence was confirmed (SUNYA, Zhejiang, CN), the pGEX-2TK construct was transformed into BL21 (DE3). The process of growth, induction and harvest was the same as that of scFv-Fc. The cell pellet team was resuspended in lysis buffer (50 mM Tris-HCl pH8.0 5 mM EDTA and 1.5 mM NaCl, phenylmethylsulfonyl fluoride). Add lysozyme (Solarbio, Beijing, CN) to a final concentration of 500 μg/mL, incubating for 30 min at room temperature and centrifuging at 12000 rpm for 30 seconds. Discard the supernatant to remove the periplasm protein. Add RIPA IV (Sangon, Shanghai, CN) and incubate on ice for 10 min, vibrating the tubes for several times during the incubation. The cell extract was clarified by centrifugation at 10000 rpm for 10 min.
Purification of scFv-Fc
The protein was purified by immunoprecipitation (IP) using anti‐DYKDDDDK G1 affinity resin (GenScript, Nanjing, CN). Resuspend the resin to form a uniform slurry and transfer 40 μL of the slurry into a 1.5 mL vial. Add 500 μL TBS into the vial and gently mix the resin, centrifuge at 6000 rcf for 30 seconds, and remove supernatant carefully. Repeat this step for three times. Add 1 mL supernatant of the cell lysate to the washed resin. Mix by end‐to‐end rotation on a tube rotator overnight at 4°C. To remove the nonspecific binding segment, centrifuge at 6000 rcf for 30 seconds, carefully remove supernatant and add 500 μL TBS to wash the resin three times. Remove as much supernatant as possible without disturbing the resin, add 120 μL of acid elution buffer (0.1 M glycine HCl, pH 3.5) into the washed resin and use a wide bore pipette tip to gently resuspend the resin. Incubate at room temperature for 5 minutes, mix gently by tapping the tube once or twice during the incubation period. After incubation, centrifuge at 6000 rcf for 30 seconds. Carefully transfer supernatant into a new vial containing 5 μL 1 M Tris, pH 9.0 for further application.
Optimum Concentration of IPTG for Expressing
The transformed cells were grown until the OD600 reached 0.6, at which time protein expression was induced by a gradient from 0 to 2 mM of IPTG. The cells were then incubated at 30°C for an additional 24 h.
Optimum Induction Time for Expressing
The transformed cells were grown until the OD600 reached 0.6, at which time protein expression was induced by 2 mM IPTG. The cells were then incubated at 30°C for a gradient from 0 to 24 h. For negative control, none inducer was introduced.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS–PAGE)
The cell lysate or purified protein was mixed 1:1 (v:v) with 2× loading buffer (TIANGEN, Beijing, CN), heated for 5 min at 95°C and subjected to SDS–PAGE (5% stacking gel, 8% separating gel). Gels were run at 110V for 15vmin and then at 160V for ~50 min in running buffer.
Coomassie Staining
Following SDS–PAGE, gels was stained with Coomassie brilliant blue (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% acetic acid) for 2 h at room temperature. Destain in destaining buffer (5% ethanol, 10% acetic acid) overnight.
Western Blotting of Proteins
Following SDS–PAGE, samples were transferred to nitrocellulose membrane (Merck) by semi-dry transfer (Bio-Rad). Membranes were blocked 1 h at room temperature in 5% (w/v) nonfat milk in TBST, probed with a rabbit anti-FLAG antibody (Abcam) overnight at 4°C, washed with TBST, probed with a goat anti-rabbit HRP-conjugated antibody (Sangon) for 1 h at room temperature, washed with TBST, and subsequently imaged using ChemiDoc (Bio-Rad).
Particularly, the blocked membranes were directly probed with a rabbit anti-mouse HRP-conjugated antibody (Sangon) as the ZZ region could bind to the secondary antibody.
Co-immunoprecipitation
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.
Cell Culture
MDA-MB-453 and MDA-MB-231 cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2 at 37°C. All cells were passaged at 80% confluency.
Targeted Cell Uptake Evaluation of scFv-Fc Using Flow Cytometry
MDA-MB-453 and MDA-MB-231 cells at 80% confluence were collected by centrifugation after treatment using 0.5% trypsin-EDTA and resuspended in 50 μL PBS. Then, the cells were washed thrice with PBS, at 400 g for 5 min. Next, the cells were incubated (4°C in dark) by scFv-Fc (or goat anti-rabbit IgG H&L (HRP) as isotype control), rabbit anti-DDDDK tag (binds to FLAG® tag sequence) antibody, goat anti-rabbit IgG H&L (Alexa Fluor® 488) and Fixable Viability Dye eFluorTM 450 (eBiosciencTM), during each incubation the cells were washed thrice with PBS, centrifuge at 400 rcf for 5 mins. In the end, the cells were resuspended in 300 μL PBS, and the Alexa Fluor® 488 and Fixable Viability Dye eFluoTM 450 fluorescence of the cells was immediately analyzed using a flow cytometer (CytoFLEX, Beckman Coulter Inc., USA).
Culture of Magnetotactic Bacteria
Magnetotacitc bacteria is a general term refers to a group of species. Here, we choose Magnetospirillum gryphiswaldense strain MSR-1 (BCRC17316) as our target. MSR-1 was purchased from Beaver County Rehabilitation Center(Taiwan, China).
The culture medium(BCRC medium no.611) of M. Gryphiswaldense MSR-1 is prepared as follows.
| Magntospirillum medium | |
|---|---|
| KH2PO4 | 0.68g |
| Na-thioglycolate | 0.05g |
| L(+)-Tartaric acid | 0.37g |
| Succinic acid | 0.37g |
| Trace elements solution | 5ml |
| Resazurin | 0.5mg |
| Distilled water | 1000ml |
| NaNO3 | 0.12g |
| Na-acetate | 0.05g |
| Vitamin solution | 10ml |
| Fe(III) quinate solution | Hyundai |
| Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N2 gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add. | |
| Trace elements solution | |
|---|---|
| Nitrilotriacetic acid | 1.5g |
| MgSO4·7H2O | 3g |
| MnSO4·2H2O | 0.5g |
| NaCl | 1g |
| CaCl2·2H2O | 0.1g |
| ZnSO4·7H2O | 0.18g |
| CuSO4·5H2O | 0.01g |
| Kal(SO4)2·12H2O | 0.02g |
| H33BO3 | 0.05g |
| Na2MoO4·2H2O | 0.01g |
| Distilled water | 1000mL |
| FeSO4·7H2O | 0.1g |
| CoSO4·7H2O | 0.18g |
| NiCl2·6H2O | 0.025g |
| Na2SeO3·5H2O | 0.3mg |
| Na2WO4·2H2O | 0.4mg |
| Trace elements solution:First dissolve nitrilotriacetic acid adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (with KOH). | |
| Vitamin solution | |
|---|---|
| Biotin | 2mg |
| Folic acid | 2mg |
| Pyridoxine-HCl | 10mg |
| Thiamine-HCl·2H20 | 5mg |
| Riboflavin | 5mg |
| Vitamin B12 | 0.1mg |
| p-Aminobenzoic acid | 5mg |
| Lipoic acid | 5mg |
| Distilled water | 1000mL |
| Nicotinic acid | 5mg |
| D-Ca-pantothenate | 5mg |
| Dissolve components in the order given. Adjust pH to 6.75 with NaOH and boil medium for 1 min. Purge medium with N2 gas for 10 min. Under the same atmosphere, anaerobically fill tubes to 1/3 of their volume and seal. Autoclave at 121℃ for 15 min. Before inoculation, add. | |
| Ferric quinate solution, 0.01 M | |
|---|---|
| FeCl3·6H2O | 0.45g |
| Quinic acid | 0.19g |
| Distilled water | 100mL |
| Ferric quinate solution:Dissolve and autoclave at 121℃ for 15 min. | |
In the incubation period, aerobic environment is conducive to the growth of M.gryphiswaldense strain MSR-1.However, MicroAeroPack or AnaeroPack is needed for fermentation period. The MicroAeroPack or AnaeroPack was purchased from Mitsubishi Gas Chemical(Tokyo, Japan).
Bacterial PCR of MSR-1
Because magnetotactic bacteria grew too slowly to form a visible colony, we tent to demonstrate that it was truly grown in the medium. The liquid medium was centrifuged at 12000 rpm for 10 minutes and the sediment was used for bacterial PCR using specific primers designed from the 16srDNA of Magnetospirillum gryphiswaldense, E.coli and Agrobacterium were used in the bacterial PCR as the negative control.
References
[1]. Ahmadzadeh, M., Farshdari, F., Nematollahi, L., Behdani, M., & Mohit, E. (2020). Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity. Molecular biotechnology, 62(1), 18–30. https://doi.org/10.1007/s12033-019-00221-2
