E. coli expression of a soluble, active single-chain antibody variable fragment containing a nuclear localization signal
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Updated: Jun 24, 2026
Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Published on: October 15, 2016
Hairong Xiong1, Shuyi Li, Zhanqiu Yang
1Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA.
Abstract:
Single-chain antibody variable fragment (scFv) proteins consist of an antibody heavy chain variable sequence joined via a flexible linker to a light chain variable sequence. Prior work has shown that ScFv 18-2 binds the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and sensitizes cancer cells to radiation following nuclear microinjection. A potential clinical delivery strategy is based on modification of the scFv so that it can be taken up into cells and imported to the nucleus. This will require development of an expression system for a nuclear localization signal (NLS)-tagged scFv derivative. We found, however, that addition of the highly basic NLS severely compromised expression in the host-vector system used for the parental scFv. After testing a variety of host strains, fusion partners, and NLS sequences and placements, successful expression was obtained with a construct containing a stabilizing N-terminal maltose binding protein tag and a single, optimized, C-terminal NLS moiety. Amylose affinity-purified ScFv 18-2 NLS protein was stable to storage at 4 degrees C in the presence of glycerol or trehalose, bound selectively to an epitope peptide, and was cleavable at an engineered Factor Xa protease site. Following lipid-mediated uptake into cultured cells, NLS-tagged ScFv 18-2, unlike the parental ScFv 18-2, localized predominantly in the cell nucleus.
Insights
Researchers developed a nuclear localization signal (NLS)-tagged single-chain variable fragment (scFv) for cancer therapy. This modified scFv successfully entered cell nuclei, overcoming previous expression challenges for targeted radiation sensitization.
Area of Science:
- Molecular Biology
- Biotechnology
- Cancer Research
Background:
- Single-chain variable fragment (scFv) proteins are engineered antibodies.
- ScFv 18-2 binds DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and sensitizes cancer cells to radiation.
- Nuclear delivery of scFv is a potential strategy for enhanced cancer therapy.
Purpose of the Study:
- To develop a nuclear localization signal (NLS)-tagged scFv derivative for intracellular delivery.
- To overcome expression challenges associated with NLS-tagged proteins in host-vector systems.
- To achieve nuclear localization of scFv 18-2 for potential therapeutic applications.
Main Methods:
- Engineered NLS-tagged scFv 18-2 constructs with various fusion partners, host strains, and NLS sequences/placements.
- Utilized a stabilizing N-terminal maltose binding protein tag and a C-terminal NLS moiety.
- Purified the NLS-tagged scFv using amylose affinity chromatography and assessed protein stability and binding.
Main Results:
- Successful expression of NLS-tagged scFv 18-2 was achieved with an optimized construct.
- The purified protein was stable, selectively bound its target epitope, and was cleavable.
- Lipid-mediated delivery resulted in predominant nuclear localization of NLS-tagged scFv 18-2 in cultured cells.
Conclusions:
- An optimized NLS-tagged scFv 18-2 expression system was successfully developed.
- The NLS-tagged scFv demonstrated effective nuclear localization after cellular uptake.
- This NLS-tagged scFv represents a promising tool for targeted nuclear delivery in cancer therapy.
