Difference between revisions of "Part:BBa K4477013"

Revision as of 02:35, 14 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, which is found within the lipid core of oxidized low density lipoprotein (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 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.

Figure 1. Plasmid map.

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).

Characterization

BioBricking

We successfully BioBricked our McPC603-encoding device.

Figure 2. Restriction Digest Verification of McPC603 Plasmid Construct. (A) Virtual digest of pSB3K3 + McPC603 with HindIII and NotI on Benchling yields three bands of sizes 2.2 kb, 1 kb, and 0.4 kb. (B) L: Quickload Purple 1 kB Plus Ladder NEB. 5: McPC603 colony digested with HindIII and NotI, 2.2 kb and 1 kb. 7: pSB3K3+mRFP with NotI, 2.5 kb and 1.2 kb. 8: pSB3K3+mRFP cut with HindIII, 2.7 kb, 0.8 kb, 0.2 kb. 9: Pink McPC603 colony, 2.7 kb, 0.8 kb, 0.2 kb. (C) 5: McPC603 colony uncut, approx. 2 kb.

After transformation of the pSB3K3+McPC603 ligation product into DH5-alpha, plasmids were extracted from 6 white colonies (Lanes 1-6 (B and C)) and 1 pink colony (Lanes 9-10 (B)) for digestion analysis. The white McPC603 colony highlighted in Figure 1 (Lane 5) matched the virtual digest when cut with the same restriction enzymes. Note that although the 0.4 kb band did not appear on the gel, this was expected as fragments shorter than 0.5 kb do not show well on this percentage of gel. The uncut colony displays a single band occurring lower on the gel than the linearized plasmid, which is expected for an uncut plasmid and indicates the plasmid preparation occurred successfully. The enzyme cutter controls we ran on pSB3K3+mRFP in lanes 7, 8, and 9 also showed expected bands.

The successful results of the verification gel were further supported by sequencing data. The extracted plasmid was found to be 97.41% similar to the theoretical McPC603 plasmid, indicating successful creation of our McPC603-encoding device.

Protein Expression

Figure 3. McPC603 Induction Trials. L: Bio-Rad Kaleidoscope Precision Ladder; 1: 16 °C for 6 hour; 2: 16 °C for 15 hour; 3: 16 °C for 24 hour; 4: 26.5 °C for 14 hour; 5: 26.5 °C for 15 hour; 6: Control: pSB1K3 + mRFP at 26.5 °C for 15 hour; 7: 26.5 °C for 24 hour; 8: 35.6 °C for 6 hour; 9: 37 °C for 15 hour; 10: 37 °C for 24 hour. Starred bands are discussed in text.

Samples of McPC603 (expected weight: 28.71 kDa) were induced at 1mM IPTG at varying temperatures and incubation time. Bands were observed at the expected molecular weight, suggesting that McPC603 is indeed being expressed by the bacteria carrying our McPC603-encoding device.

Sequence and Features