Part:BBa_K5234001
anti-CA125-scFv
This part encodes an antibody, anti-CA125 scFv.
Usage and Biology
by BWYA 2024
Our project addresses the critical need for enhanced risk screening and early detection of gynecological diseases. An array of biomarkers should be studied and included to tackle this challenge, and as a proof-of-concept, one of the biomarkers we studied, is CA125.
CA125 is mostly known as an indicator of ovarian cancer, while at the same time, its concentration shows abnormal elevations in many gynecological diseases, such as Endometriosis, Endometrial cancer, Adenomyosis, and etc. The diseases indicated by CA125 covers a wide range of population group.
To detect the biomarker, we intended to utilize the antigen-antibody binding mechanism. We believe this approach will lead to portable, non-invasive, and thus user-friendly gynecological care products.
Producing full-length antibodies is very challenging. To address this challenge, we opted for single-chain variable fragment (scFv) antibodies. They are minimal antigen-binding fragments, approximately 25 kDa. They are composed of the variable domains from both the heavy (VH) and light (VL) chains bridged by a linking sequence. The scFv antibodies can be more easily expressed in simple prokaryotic bacteria.
This part encodes a specific scFv, anti-CA125-scFv. We selected humanized 4H11 (h4H11) antibody against the specific CA125 ectodomain. The ability of the 4H11 to recognize CA125 was proven experimentally by previous works in Fluorescence Activated Cell Sorting (FACs) [1].
We expressed the scFv with a strong T7 promoter, aided with a lac operator (LacO), all carried on a pET28a vector. The plasmid was transformed into E. coli (BL21) for expression. The bacteria strains were cultured in LB medium with proper antibiotics till the OD600 reached 0.6 - 0.8, and then 0.3 mM IPTG was added to induce expression. Incubate the bacteria culture overnight at 20°C with a 200 rpm. Transfer the bacterial culture to a centrifuge tube and subject it to ultrasonication at a power setting of 30W for 10 minutes. Following ultrasonication, centrifuge at 12,000 rpm for 5 minutes, then carefully aspirate and collect the supernatant into a fresh centrifuge tube. Resuspend the precipitate with 1 mL of 20 mM Tris-HCl buffer. Perform SDS-PAGE validation assays on both the supernatant and the precipitate.
Fig. 1: SDS-Page run verified the successful expression of anti-CA125-scFv at about 28kDa.
In order to use the antibodies for lateral flow assay implementation, protein extraction and purification is necessary. We experimented with conditions to purify the protein using Tris-HCl buffer, guanidine hydrochloride and imidazole denaturing solution, as well as a column packed with nickel resin that resists denaturation. The fermented bacteria were resuspended, sonicated, and the supernatant and precipitation were separated, with the precipitation being redissolved. Samples of both the supernatant (sup. ) and the precipitation (prec.) were taken for analysis.
Fig. 2: SDS-Page run of the supernatant, precipitate, and the elution from purification runs.
To further enhance protein solubility and expression, we considered using the Shuffle T7 strain, which is designed to improve the solubility of recombinant proteins. Shuffle T7 is genetically engineered for efficient disulfide bond formation in the cytoplasm, making it ideal for proteins requiring proper folding.
Fig. 3: SDS-Page run compares the expression of anti-CA125-scFv in BL21 and Shuffle T7 strains. The results indicate the expression in supernatant and precipitates are comparable in Shuffle T7, an improved results from BL21.
Solubilisation of Inclusion Bodies
The previous experimental results showed that after the production of anti-CA125 scFv, a lot of it existed in the form of inclusion bodies. In order to solubilize the protein within the inclusion bodies, we attempted purification with non-denaturing agents.
Experiment procedure:
1、Collect cell pellets after bacteria culture fermentation, resuspend the pellets with 20 mM Tris-Cl, and lysis with ultrasonication;
2、Separate supernatant and precipitations, and dissolve the precipitations with non-denaturing agents.
3、Pass the above re-dissolved solution through Ni column and wash with 5 column volume.
4. Elute with 2 to 3 column volumes of elution buffer.
5. Collect the protein eluate and concentrate it.
Non-denaturing solvent/wash buffer:
1% (w/v) N-lauroyl sarcosine (NLS) [2] ，300 mM NaCl ，50 mM Tris-Cl ，pH 8.0
Elution Buffer：
0.1% (w/v) NLS ，300 mM NaCl, 50 mM Tris-Cl ，300 mM Imidazole ，pH 8.0
The SDS-Page run after purification and elution clearly shows a clear band for the anti-CA125 scFv and it proves the anti-CA125 scFv can be properly solubilized. We estimated the production quantity of this scFv to be 4 mg/L.
Binding Interaction Verification
To verify whether the designed scFv antibodies can bind to their corresponding antigens, we used a simpler method, native PAGE, instead of more complex techniques such as ELISA or co-immunoprecipitation. We incubated the anti-CA125 scFv antibody with the purchased commercial CA125 antigen protein at room temperature for 2 hours and then performed native-PAGE under non-denaturing conditions.
In the native PAGE results, it is evident that the position of the bands shifted after incubation of the CA125 antigen protein with our anti-CA125 scFv. The CA125 antigen protein has an original size of approximately 80 kDa. Due to native PAGE being a non-denaturing electrophoresis, the protein is not linearized and appears at a position slightly higher than its expected size.
The anti-CA125 scFv, on the other hand, is a much smaller protein, with a size of about 28 kDa. We believe that due to the binding between the two, the CA125 antigen protein was pulled down by the smaller scFv, causing the shift in band position. Therefore, we infer that our designed scFv can effectively bind to the target antigen.
Reference
[1] Lee, K., Perry, K., Xu, M. et al. Structural basis for antibody recognition of the proximal MUC16 ectodomain. J Ovarian Res 17, 41 (2024).
[2] Şahinbaş, D., & Çelik, E. (2023). Enhanced production and single-step purification of biologically active recombinant anti-IL6 scFv from Escherichia coli inclusion bodies. Process Biochemistry, 133, 151-157.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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