Part:BBa_K3593012

Long description

This composite part is meant to express the anti-α-amanitin scFv gene under T7 promoter in insoluble form. scFv is a type of single-chained antibody, short for single-chain fragment variable. It can be produced by standard molecular cloning and protein production techniques with bacteria, is a great improvement from traditional antibodies that need animals cells for production. This scFv gene acts as an antibody against α-amanitin, a subtype of the toxin found in genus Amanita as well as some other mushroom species. This scFv posed relatively good specificity with an IC50 of 77.48 ng/mL and IC15 of 1.91 ng/mL using ic-ELISA. 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.

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 101
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Design

To obtain the pure sample of scFv for further test and control, we constructed this part to obtain purified scFv. Because the inclusion body has almost all pure protein in it, so we can get a pure sample. However, the inclusion bodies are not functioning form of our scFv.

The plasmid map showing the structure of pET28b-scFv-HisTag's structure

Experiment data

BBa_K3593012 was characterized in the following experiments:
-Protein expression
-Inclusion body extraction

Protein expression

This scFv gene with 6*His-tag at its C-terminal and was cloned into pET28b expressed in E.coli BL21(DE3) by LB 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 200RPM. Whole culture were collected after 24h incubation after induction by centrifuging, lysed by ultrasonic and lysozyme lysis. Supernatant and cell pellet are collected by centrifugation. When using SDS-PAGE to observe the lysed pellet, target bands were shown at the size of 28kDa while the lysed supernatant showed no results. It was concluded that inclusion bodies have formed during protein expression. Inclusion body extraction was therefore applied.

Fig.1 The SDS-PAGE showing the expression of scFv in periplasm and inclusion body.

Inclusion body extraction

The pellet collected from ultrasonic lysis was resuspended in PBS and lysed again. By multiple times of centrifugation and washing, and the addition of resuspension buffer, pellet that is ready for extraction can be collected by a final centrifugation. Dissolution buffer containing 8M urea was used to dissolve the pellet. With the analysis of SDS-PAGE, it was confirmed that scFv inclusion body was present and extracted successfully.

Fig.2 The SDS-PAGE showing that the solublization of scFv inclusion bodies and pelB scFv inclusion bodies.

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