Part:BBa_K5234000
anti-IL-6-scFv
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 an anti-IL-6 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 IL-6.
IL-6 is a common inflammation marker. In fact, gynecological inflammation is significantly more common than cancers in female patients. Such inflammations and infections are also hard to cure and often transform into chronicle conditions, posing a long-term burden on females. Besides, IL-6 is not only associated with inflammations but also indicative of various other conditions, including some cancers.
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-IL-6-scFv. We selected the antibody from a newly published work. In the work, Biozzi mice were immunized with IL-6 emulsified in sodium chloride and Freund's adjuvant, followed by booster injections, and a final intraperitoneal boost. Splenocytes were harvested and fused with mouse myeloma cells to create hybridomas, which were screened by ELISA. Positive clones were cloned and injected into mice for ascites production. RNA was extracted from hybridoma cultures containing about 10^7 cells. The first-strand cDNA was synthesized from 1 μg RNA by reverse transcription, and thus the antibody was identified. [1] [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 27kDa.
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.
Reference
[1] 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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