Part:BBa_K1391100:Experience

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Applications of BBa_K1391100

This is a fusion of a beta-amyloid specific variable region on a human IgM heavy chain with a membrane localization tag that causes it to be sent to the ER and eventually the membrane with other parts. The heavy and light chain stick to and extracellular antigen (beta-amyloid) and cause the B-Cell Receptor (BCR) complex to dimerize. The IgM Heavy chain, IgM light chain, CD79A subunit, and CD79B subunit require processing in the ER and must all be expressed in order to membrane localize instead of being retained in the ER by quality checking mechanisms. Binding to the antigen (beta-amyloid) causes two BCR complexes to dimerize. This allows Lyn to phosphorylate the CD79 heterodimer which causes the recruitment of Syk. As a warning, recruited Syk can cause cross talk with native cell signaling pathways as can other proteins. Care must be taken to check protein interactions and point mutate Syk if needed. We attach a protease (TEV protease) to Syk and a transcriptional activator (Gal4-VP16) to CD79 using a cleavage site (TEV cleavage site). When The BCR is activated by our antigen, Syk-TEVp cleaves the cleavage sit and releases Gal4-VP16 which can activate a synthetic system. The variable regions of the heavy and light chains can be switched for the variable regions of another monoclonal antibody with known cDNA or peptide sequence to change which antigen causes activation. It is important to make sure an appropriate localization tag is attached to the new heavy and light chains. The tag must be cleaved correctly, leaving no residue and removing no variable region peptides. This part is under a constitutive hEF1a promoter for mammalian cells.

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