In Silico Generation Of Scfv Antibodies Against Salmonella Enterica Ser. Typhi (S. Typhi) Tolc Protein
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Date
2015-07
Authors
Leong, Siew Wen
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Abstract
Typhoid fever is a global health concern especially in developing and under-developed regions such as Southeast Asia, India, Africa and South America. The latest comprehensive study on the spread of the disease estimated that 21.7 million cases of typhoid fever occur annually. Subsequently, 1 % of the cases ended in fatality. Therefore, rapid diagnostics play an important role particularly in rural places for efficient disease control and treatment. In recent years, a novel approach in creating de novo binders for protein targets from non-related scaffolds has been developed, creating many possibilities for therapeutics and also diagnostics. This de novo design approach incorporates hotspot residues onto a suitable protein scaffold, creating new binders that can recapitulate natural protein-protein interfaces. In this work, the de novo design approach is used to create binders in scFv format for Salmonella enterica serovar Typhi (the causative agent for typhoid fever) TolC protein. The TolC-like protein has been found to be antigenic and specific for the pathogen and was subsequently developed into dot enzyme immunoassay diagnostic kit, Typhidot®. Molecular dynamics simulation was first performed on a homology model of the TolC protein in a POPE membrane lipid bilayer model. The protein showed flexibility in the extracellular loops and C-terminal regions. Clustering analysis on the molecular dynamics trajectories converged upon the structure at 13.12 ns as the central structure. Thus this most dominant structure was used as the target for single-chain variable fragment (scFv) binder design. Potential hotspot residues capable of binding to the target protein with high affinity were identified by docking “disembodied” amino acid residues against the target surface. Next, scFv scaffolds were selected from the RCSB Protein Data Bank (PDB) to harbor the computed hotspot residues.
The design process incorporated the hotspot residues onto the scFv scaffold complementarity determining regions (CDRs), and redesigned neighbouring residues to optimize the binding affinity. The designs have interface characteristics similar to natural antibody-protein complexes in terms of binding energy, shape complementarity and interface surface area. Five high potential structures with high binding energy have been selected and analyzed. They should be further examined at in vitro to distinguish their potential prior to the development of antigen detection agents for typhoid fever diagnostics.
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Keywords
Typhoid Fever