Difference between revisions of "Part:BBa K4477012"

Revision as of 13:58, 12 October 2022

IK17 (anti-oxLDL) scFv - complete expression cassette

Overview

This sequence codes for a short chain variable fragment (scFv) derived from the anti-oxLDL IK17 antibody. Specifically, this scFv binds to MDA-modified apoB, or the protein component of oxLDL (1). OxLDL stands for oxidized low density lipoprotein (oxLDL), and it is a biomarker of many inflammatory diseases such as atherosclerosis, so antibodies able to target oxLDL could enable potential detection and therapeutic applications for such diseases.

This sequence is one of three antibody sequences designed by Virginia 2022 to encode antibodies that bind to oxidized LDL (OxLDL). It is a composite part with a T7 promoter, E. coli-optimized RBS, coding sequence, and terminator. A strong T7 promoter is located at the 5' end of the sequence to facilitate IPTG induction. The RBS was codon-optimized using the Salis RBS calculator. The CDS is, of course, derived from the antibody IK17, and codes for the following in sequential order: IK17 variable heavy chain, a (G4S)3 linker sequnece, and IK17 variable light chain. The (G4S)3 linker sequence was chosen because it was characterized to enhanced protein expression of recombinant antibodies (2). Note that the protein resulting from this sequence will only fold correctly in SHuffle E. coli, or a similar oxidizing-cytoplasm strain, as there are disulfide bonds present in its structure. Non-illegal restriction sites were included between each part for modularity. Using these restriction enzymes, parts can be swapped to vary translational rates and protein expression.

Usage and Biology

IK17 is a human monoclonal IgG Fab antibody that is a strong binder to the modified-apoB protein of malondialdehyde-modified-oxLDL (MDA-LDL) and Copper-LDL(Cu-LDL) and does not bind significantly to other antigens. The dissociation constant is 3.7 x 10^(-8) mol/L.

Different competitors that may affect the binding of IK17 to MDA-LDL were evaluated. IK17 bound extensively to the apoB protein of Cu-OxLDL, MDA-LDL, protein moieties of MDA-HDL, but did not bind to the protein moieties of native LDL or HDL. IK17 also inhibits the ability of macrophages to uptake oxLDL, inhibits apoptotic cells from being phagocytosed by macrophages. IK17 binds to atherosclerotic lesions in vivo.

IK17 may be used for therapeutic purposes to block the uptake of oxLDL by macrophages, which would reduce the amount of foam cells in the atherosclerotic lesion and delay the buildup of plaque and progression of atherosclerosis (3).

Sequence and Features