Part:BBa_K4923003
Anti-PSMA - complete expression cassette
Overview
This sequence codes for the hybrid antibody Anti-PSMA. It is derived partially from J591, a monoclonal antibody that specifically targets PSMA. PSMA (Prostate-specific Membrane Antigen) is a protein that is primarily expressed in the cells of the prostate gland. It acts as a biomarker which is located on the outside of prostate cancer cells. Antibodies able to target PSMA could facilitate potential detection and therapeutic applications for diseases mainly prostate cancer.
Due to the presence of a promoter in our corresponding plasmid, which will be introduced shortly, there is no necessity for an additional promoter in this antibody. Thus, the composite part includes just two RBS(Ribosome Binding Sites) and one terminator, resulting in the simultaneous transcription of both the coding sequences (CDS) for the light chain and the heavy chain. The initial segment of the sequence contains a strong Ribosome Binding Site (RBS) known as BBa_J61100, available in the Registry. It is then followed by the light chain of Anti-PSMA, which consist of both variable region and constant region and can be found in the Registry as part BBa_K4923001. Positioned between the light chain and heavy chain is a scar, along with the strong RBS BBa_J61100. The scar consists of a stop codon TAG and a sequence resembling the BioBrick scar, but with slight variations at the beginning and end, lacking the T and G. Subsequently, the heavy chain of Anti-PSMA follows, again consisting of both the variable region and constant region, identified as BBa_K4923002 in the Registry. Finally, a two-way terminator BBa_B0015 appears, serving the purpose of preventing RNA polymerases from reading beyond the composite part into the backbone and inhibiting the transcription of any genes in the backbone into the composite part. Both the light and heavy chains of a full-length antibody is shown in the diagram below.
In terms of the plasmid we used, two plasmids were selected to transform this antibody gene. The first plasmid, pET-21b, was chosen due to its high replication rate and the presence of the T7 promoter. The second plasmid, pSB3K3, was sourced from the Registry. We initially performed two groups of experiments using two plasmids simultaneously and transformed them. However, during gel electrophoresis, the observed band position did not align with the expected position for pSB3K3 due to the low expression level of protein, so we eventually ceased the experiment with pSB3K3 and proceeded solely with pET-21b for further experimentation.
pET-21b possesses multiple sites for digestion. We used two digestion enzymes EcoRI and HindIII to cut open the plasmid and insert the antibody gene. The complete map of the plasmid is illustrated in the figure below.
Usage and Biology
J591 antibody was developed by Memorial Sloan Kettering Cancer Center and was initially described in scientific publications in the early 2000s.
J591 antibody is completely derived from the cells of Mus Musculus, which is a kind of mouse. This monoclonal antibody can be utilised in targeted therapy approaches for prostate cancer. It specifically binds to and targets PSMA-expressing cancer cells, which are commonly utilised with the use of radioactive isotopes to selectively destroy the cancer cells.
J591 antibody is currently being tested for its ability to become personalised medicine for prostate cancer. Utilising J591-based imaging techniques, along with genetic profiling, may help identify patients who are most likely to benefit from J591-based therapies, which could lead to more accurate and tailored treatment strategies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 587
Illegal BglII site found at 2316
Illegal BamHI site found at 562
Illegal BamHI site found at 1630
Illegal XhoI site found at 676
Illegal XhoI site found at 1624 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 524
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 75
Illegal BsaI site found at 1388
Illegal BsaI.rc site found at 2149
Illegal BsaI.rc site found at 2617
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