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Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing
Alessio Lombardi1, Maria Sperandei, Cristina Cantale
1ENEA, BIOTEC-GEN Unit, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy.
Abstract:
Recombinant antibody fragments represent useful tools for cancer diagnosis and therapy. Aberrant expression of the HER2 receptor is implicated in metastatic breast and ovary cancers, two malignancies with a high prevalence in young women. In this study, we focussed on a single-chain fragment of variable antibody regions specific for HER2 (scFv800E6) that can be expressed in a functional form in the cytoplasm of Escherichia coli. ScFv800E6 was extracted from bacterial cultures following induction at different temperatures and purified. The yield of both soluble and insoluble forms was measured. We found that scFv800E6 was functional when expressed in the soluble fraction in the bacteria cytosol. In addition, scFv800E6 extracted from inclusion bodies was easily refolded and largely recovered its functionality. Thus, scFv800E6 is intrinsically capable of efficient and functional folding in a reducing environment and represents one of the few described antibody fragments with a framework well adapted for cytoplasmic expression.
Insights
This study shows that a recombinant antibody fragment targeting HER2 (scFv800E6) can be effectively produced in E. coli cytoplasm. The fragment maintains functionality whether soluble or refolded from inclusion bodies, aiding cancer therapy development.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Recombinant antibody fragments are valuable for cancer diagnosis and therapy.
- Aberrant HER2 receptor expression is linked to metastatic breast and ovarian cancers, prevalent in young women.
Purpose of the Study:
- To investigate the functional expression of a HER2-specific single-chain variable fragment (scFv800E6) in the cytoplasm of Escherichia coli.
- To assess the yield and functionality of scFv800E6 in both soluble and insoluble fractions.
Main Methods:
- Expression of scFv800E6 in E. coli cytoplasm under various induction temperatures.
- Extraction and purification of scFv800E6 from bacterial cultures.
- Measurement of soluble and insoluble scFv800E6 yields.
- Assessment of fragment functionality after extraction and refolding.
Main Results:
- scFv800E6 was successfully expressed and functional in the soluble fraction of the bacterial cytosol.
- scFv800E6 extracted from inclusion bodies could be easily refolded, largely retaining its functionality.
- The antibody fragment demonstrated efficient and functional folding in a reducing environment.
Conclusions:
- scFv800E6 is well-suited for cytoplasmic expression in E. coli due to its intrinsic folding capabilities.
- This antibody fragment represents a promising tool for developing novel cancer diagnostics and therapeutics targeting HER2.
