Difference between revisions of "Part:BBa K4477013"

Revision as of 13:08, 12 October 2022

McPC603 anti-oxLDL scFv - complete expression cassette

Overview

This sequence codes for a short chain variable fragment (scFv) derived from the anti-phosphocholine McPC603 antibody. McPC603 targets a chemical group called oxidized phosphocholine found within the lipid core of oxidized low density lipoprotein, or oxLDL. OxLDL is a biomarker of many inflammatory diseases such as atherosclerosis, so antibodies able to target oxLDL such as this one could enable potential detection and therapeutic applications for such diseases. The sequence codes for the following in sequential order: McPC603 variable heavy chain, a (G4S)3 linker sequence, and McPC603 variable light chain.

This sequence is one of three antibody sequences designed by Viginia 2022 to bind specifically to oxidized LDL (OxLDL). It is a composite part, complete with a T7 promoter (BBa_I712074), E. coli-optimized ribosomal binding site (RBS) (BBa_K4477009), coding sequence (CDS) (BBa_K4477004), and terminator (BBa_B0014). A strong T7 promoter was included on the 5' end of the sequence, enabling IPTG induction. The RBS was designed using the Salis RBS calcuator for expression in E. coli. The coding sequence is derived from the antibody McPC603 (1), which binds to oxidized phosphocholine on OxLDL. The variable heavy and light chain of McPC603 are connected by a (G4S)3 linker sequence (2). This linker sequence was chosen because it was characterized to enhanced protein expression of recombinant antibodies. Note that the protein resulting from this sequence will only fold correctly in SHuffle E. coli or another oxidizing-cytoplasm bacterial strain, as there are disulfide bonds present in its structure. As with other Virginia 2022 composite parts, non-illegal restriction sites were included between each part for modular design.

Usage and Biology

Our McPC603 derivative is a member of a category of antibody fragments called single chain variable fragments (scFv).

ScFvs are synthetic antibody fragments composed of the variable heavy and variable light chains of a pre-existing antibody, joined together with a linker sequence of 15-20 amino acid residues. These synthesized antibody fragments, while originally created to facilitate phage display, have been used for a wide range of laboratory purposes such as immunohistochemistry and flow cytometry.

Figure 1. Subunit composition of an scFv. Image acquired from Ahmad et al 2012 (3)

McPC603 is a monoclonal antibody that targets phosphocholine, the product that results when the polar head of a phospholipid becomes oxidized. Phosphocholine compounds are present in the lipid core of LDL, and only appear when the LDL is in an oxidized state.

McPC603 was used as an antibody model for structural analysis. McPC603 was also used as a basis for the development of E06, a therapeutic antibody for atherosclerosis, by blocking macrophages uptake of ox-LDL (4).

Sequence and Features