Difference between revisions of "Part:BBa K4477011"

Revision as of 09:37, 12 October 2022

Human/mouse anti-apoB(MDA) (anti-oxLDL) IgG - complete expression cassette

This sequence codes for a hybrid human/mouse anti-apoB(MDA) IgG antibody. It is derived partially from Orticumab, a human antibody that is currently in development as a therapeutic drug. ApoB(MDA) is a specific chemical structure found as a component of oxidized low density lipoprotein (oxLDL). OxLDL 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.

Past researchers have found success in expressing full-length antibodies in SHuffle when the heavy and light chains are arranged polycistronically; that is, they are transcribed onto the same mRNA, and then translated into separate proteins downstream (1). Thus, this composite part includes just one promoter and one terminator, which cause both the coding sequence (CDS) for the light chain and that for the heavy chain to be transcribed together. The promoter is BBa_I712074, a strong T7 promoter that allows this composite part to be expressed through IPTG induction. The terminator is BBa_B0014, a two-way terminator that not only prevents RNA polymerases from reading past the composite part into the backbone, but prevents any genes in the backbone from being transcribed into the composite part. Bracketed by the promoter and terminator are the two translational units: first, the RBS and CDS for the light chain, and then the RBS and CDS for the heavy chain. Both RBSs were designed using the Salis RBS calculator to promote optimal translation of their respective CDSs and can be found in the Registry as parts BBa_K4477006 and BBa_K4477006.

Both the light and heavy chains of a full-length IgG contain multiple subunits, as shown in the diagram below. File:IgG Subunits.jpg Diagram used under Creative Common license.

The light chain CDS encodes the light chain subunits in the following order, from upstream to downstream: variable light (VL), constant light (CL). The entire light chain amino acid sequence is identical to that of Orticumab, although codons were optimized for E. coli and to domesticate the sequence for RFC[10] and Type IIS assembly methods. The heavy chain CDS encodes the following subunits, from upstream to downstream: variable heavy (VH), constant heavy 1 (CH1), constant heavy 2 (CH2), constant heavy 3 (CH3). The VH and CH1 amino acid sequences were derived from Orticumab, while the CH2 and CH3 domains were derived from a murine (house mouse; Mus musculus) IgG heavy chain. Note that legal restriction enzyme sites were included between each subcomponent for modularity. Using these restriction enzymes, one can easily switch out the RBS to increase or decrease translational rates, for instance.

Sequence and Features