Part:BBa_K3593013

pelB-scFv(anti alpha-amatinin)-6*His
This composite part is meant to express the anti-amanitin scFv gene under T7 promoter and in soluble form then purify scFv antibody by IMAC. scFv, single-chain fragment variable, is a type of single-chain antibody. It can be produced by standard molecular cloning and protein production techniques with bacteria, are a great improvement from traditional antibodies that need animals for production. The addition of a pelB tag allows bacteria to secrete the protein to its oxidizing periplasm, preventing the formation of insoluble inclusion bodies. This scFv gene acts as an antibody against α-amanitin, a subtype of the toxin found in Amanita exitialis as well as some other mushroom species. It is meant to provide a great enhancement in toxin detection for its promising ability to be used in ELISA, enzyme-linked immunosorbent assay and LFIA, lateral flow immunoassays. This part is based on the BBa_3593011.

Background

It is very hard to produce the whole antibody molecule inside the Prokaryotes because of the aggregation of peptides that would happen to form inclusion bodies because of the reductive environment of cytoplasm, and also the need of post-translational modifications. So we discovered a single-chain fragment variable (scFv) antibody specifically targeting α-amanitin constructed by Dr.He[1] previously. scFv consists of VH and VL chain joined together by a flexible linker. These modifications enables the scFv to be expressed in E.coli, which is what we want to achieve. And this fragment can be used to construct ic-ELISA kit and LFIA bar to detect the amatoxin.

Sequences and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 164
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 54
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Design

To acquire solvable form of scFv, we have added a pelB tag to the scFv N-term to help it be secreted into the oxidizing periplasm after translation, in a solvable form that we can collect from the periplasm and the culture. Also, we added a 6*His Tag to the scFv for the purification after acquiring the crude extract of the protein by the IMAC, and also the histag plays an epitope for antibody to detect so we can use LFIA and ic-ELISA method to detect the toxin.

Fig.1 The plasmid map showing the structure of pET28b-pelB-scFv-HisTag

The cross-reactivity data and the IC 50 from the SPR sensor is mentioned in literature[1], which showed the scFv can effectively detect amatoxins because of high affinity, especially α-amanitin. For further development, we may mutate the DNA sequence of our scFV and change the amino acid sequence of the CDR groups to obtain scFv with higher affinity towards amatoxin.

Fig.2 the Cross reactivity data and the IC 50 data in the article [1]

Experiment data

BBa_K3593013 was characterized in the following experiments:
-Protein expression
-Protein purification
-ELISA measurement and analysis

Protein expression

This scFv gene with 6His-tag at its C-terminal was cloned into pET28b and expressed in E.coli BL21(DE3) by LB 100mM glucose medium growth to OD600=0.9, followed a switch of medium to 2YTS and 1mM IPTG induction. It was then raised at room temperature and shaking at 200 RPM. Whole cells were collected after induction by centrifuging, lysed by ultrasonic lysis, supernatant and cell pellet collected by centrifugation. The anti-His tag colloidal gold competitive test strips were used to verify the production of scFv, and a positive result is given. SDS-PAGE on cell lysate also verify the production of scFv.

Fig.3 Colloidal gold anti-His tag competitive test strips showing positive result by one red control line(the upper test strip), and negative result given by red colour in both test line and control lines(the lower test strip).

Fig.4 SDS-PAGE operation showing strong bands in cell pellet and lysate at the scFv target size, about 28kDa.

LB 100mM Gulcose formula/1L	10 g of Bacto-Tryptone	5 g of Bacto-Yeast extract	18 g of Glucose	5 g of NaCl	pH 7.5
2YTS medium/1L	16 g of Bacto-Tryptone	10 g of Bacto-Yeast extract	137 g of Sucrose	5 g of NaCl	pH 7.5

Protein extraction

We have used osmotic shock to extract the scFv in the periplasm, and then obtained the extract that can be used to conduct ELISA. The method we have used is as follows.
1. Centrifuge, 5000xg 10 min at 4℃ to collect the cell.
2. Resuspend the pellet in 10% original volume of 0.2 mol/L Tris–HCl, pH 8.0, 0.5 mmol/L EDTA, 0.5 mmol/L sucrose buffer.
3. Incubate the mixture on ice for 30 min.
4. Centrifuge, 12000xg 20min at 4℃, dispose the supernatant.
5. Resuspend the pellet in 2% original volume of 5mM MgSO4 buffer, incubate at room temperature for 10 min.
6. Centrifuge, 12000xg 20min at 4℃, collect the supernatant, and that is the Osmotic shock product.

Fig.5 The SDS-PAGE showing the product of osmotic shock product having target band at about 28kDa.

Protein purification

This scFv gene has 6His tag fused onto the C-terminal, and a pelB tag to transport protein into the periplasm then increase soluble expression, therefore standard Ni-NTA affinity column chromatography was used to purify. However, as pelB tag is added, the structure changes and the pelB tag blocks the His tag, so the protein’s affinity towards Ni resins are lowered. As shown in the SDS-PAGE of purified samples, W1-W4 showed target bands while elution samples showed weak bands.

Fig.6 Ni-NTA affinity column purification showing weak bands in elution samples but target bands in elusion samples.

ELISA measurements and analysis

As protein purification does not give out enough scFv for ELISA and test strip, we have used the cell lysate to conduct ELISA. As the cell lysate contains enough amount of scFv in it, and the concentration of scFv doesn't change in the same batch of protein, it is acceptable to use the cell lysate in the final product. These antibodies were used for ELISA and indirect competitive ELISA(ic-ELISA), with CBS of pH 9.6 to coat 0.1 ug/L α-amanitin to the 96-well plate. After incubating the sample with scFv together, the plate is washed with PBST and then adding mouse anti-Histag antibody-HRP into each well, incubate again and wash with PBST. After that add TMB solution to each well and color change is observed. Then we use the plate reader to read the plate at 450 nm to quantify the toxin in the sample.

Fig.7 scFv ELISA verified the binding of scFv and α-amanitin.

Fig.8 scFv ELISA testing the IC 50 of our scFv protein.

References

1. Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3
2. Expression B. Bacterial Expression, Purification, and Characterization of Single-Chain Antibodies. :1035-1046. doi:10.1385/1-59259-169-8:1035