Difference between revisions of "Part:BBa K4477012"

Revision as of 15:14, 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).

Characterization

BioBricking

We successfully BioBricked our IK17-encoding device.

Figure 1. Restriction Digest Verification of IK17 Plasmid Construct. (A) Virtual digest with PstI, XbaI, and HindIII on Benchling yields three bands at 2.2 kb, 1 kb, and 0.4 kb. (B) L: Quickload Purple 1 kB Plus Ladder From NEB. 1: IK17 uncut, 2 kb. 2: IK17 cut with XbaI, PstI, HindIII, 2.2kb and 1kb. 8: pSB3K3+mRFP cut with HindIII, 10 kb, 6 kb, and 4 kb. 9: pSB3K3+mRFP cut with PstI, 6 kb. 10: pSB3K3+mRFP cut with XbaI, 5 kb and 2.5 kb.

After transformation of the pSB3K3+IK17 ligation product into DH5-alpha, plasmids were extracted from 6 white colonies and 1 pink colony for digestion analysis. As seen in Figure 4, the uncut and cut IK17 colony plasmids seen in lanes 1 and 2 respectively produced expected results. The uncut IK17 plasmid lane showed one band below the molecular weight predicted for the plasmid if the plasmid were linearized, while the cut IK17 plasmid showed bands at 2.2 kb and 1 kb as predicted (the 0.4 kb band was not expected to appear on the gel). However, the cutter controls in lanes 8, 9, and 10 showed bands at much higher molecular weights than predicted, indicating potential incomplete digesting activity by these enzymes.

The results of the verification gel were further supported by sequencing data. The extracted plasmid was found to be 97.60% similar to the theoretical VHH plasmid, indicating successful creation of our IK17-encoding device.

Protein Expression

Figure 2. SDS-PAGE analysis of IK17 Induction. T represents total lysate, S represents soluble fraction, control represents pSB1K3 + mRFP. L: Bio-Rad Kaleidoscope Precision Ladder; 1: IK17 T; 3: control T; 4: IK17 S; 6: control S. Note the presence of a band at 28 kb (the expected weight of IK17) in Lane 1. Lanes 2 and 5 held samples for a different device and will not be discussed.

Samples of IK17 (expected weight: 28.69 kDa) were induced at 1mM IPTG. Bands were observed at the expected molecular weight, suggesting that IK17 is indeed being expressed by the bacteria carrying our IK17-encoding device.

Sequence and Features