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Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides
Wei Hu1, Qiuhong Xie1,2,3, Hongyu Xiang1,2,3
1School of Life Science, Jilin University, Changchun, Jilin 130012, China.
Abstract:
The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.
Insights
Engineered oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragments (scFvs) show enhanced binding activity. Fusing LOX-1 binding heptapeptides to scFvs improves therapeutic delivery for atherosclerotic diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragments (scFvs) are crucial for targeted delivery in atherosclerotic disease therapy.
- Current applications are hindered by the low antigen affinity of existing scFvs.
Purpose of the Study:
- To enhance the LOX-1 binding activity of anti-LOX-1 scFvs.
- To investigate the impact of fusing LOX-1 binding heptapeptides on scFv efficacy and stability.
Main Methods:
- Construction of a 3D model of anti-LOX-1 scFv and docking with LOX-1 protein.
- Design and synthesis of scFv/heptapeptide fusions at various positions (N-terminus, C-terminus, linker).
- Expression in Brevibacillus choshinensis, purification, and characterization of fusion proteins (binding activity, stability).
Main Results:
- Fusion of LOX-1 binding heptapeptides, particularly at the N-terminus, significantly increased LOX-1 binding activity.
- The enhanced binding was achieved without compromising the structural characteristics, thermal stability, or serum stability of the scFvs.
- Computational analysis identified distinct LOX-1 binding interfaces for the scFv and peptide components.
Conclusions:
- N-terminally fused scFv/peptide constructs represent a promising strategy to improve LOX-1 targeting efficacy.
- These enhanced scFvs hold potential for increased application in LOX-1-based diagnostics and therapeutics for atherosclerotic diseases.
- The study provides a foundation for developing more effective targeted therapies for cardiovascular conditions.
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