Part:BBa_K4477012

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, the sequence codes for the following in sequential order: IK17 variable heavy chain, a (G4S)3 linker sequnece, and IK17 variable light chain.

This sequence is one of two antibody sequences designed by Virginia 2022 to 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 derived from the antibody IK-17. This binds to MDA-modified apo-B, or the protein component of OxLDL (1). The variable heavy and light chain of IK-17 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, 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 576
  
• 1000
  COMPATIBLE WITH RFC[1000]