PSF-CMV-HUIGG1 HC - HUMAN IGG HEAVY CHAIN PLASMID

PSF-CMV-HUIGG1 HC - human IgG heavy chain expression plasmid contains the human IgG heavy chain constant region and has been designed to allow the seamless fusion of variable antibody fragments to create full length heavy chain expression constructs. The plasmid vector has been engineered to contain a BseRI restriction enzyme site upstream of the constant domain that when cleaved produces an overhang of the first two bases of the first codon of the constant region. This allows variable antibody fragments to be cloned upstream to create full length antibody genes. We also provide plasmids for human Kappa and Lambda light chain expression (termed pSF-CMV-HuKappa LC and pSF-CMV-HuLambda LC repectively). We also have a range of antibody expression plasmids for mouse and rat IgG heavy and light chains.

Promoter Expression Level: This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture.

PSF-CMV-HUIGG1 HC - human IgG heavy chain plasmid encodes a heavy chain constant region in the main multiple cloning site positioned so that it can be cleaved to produce an overhang that allows seamless fusion with a heavy chain variable region from any antibody. This allows you to create full length heavy chain antibody genes with no cloning scars.

To enable this immediately upstream of the constant region coding sequence there is a BseRI restriction site. This is a type-IIS restriction enzyme that binds in one position (CAGCAG) and then cleaves a specific number of nucleotides away from the binding site regardless of the sequence at the cleavage point. We use this site in all of our antibody expression cassettes in the same position. In this plasmid cutting with BseRI will result in an overhang consisting of the first two nucleotides of the first codon of the constant region. This means that any variable region with the same overhang at its 3 prime end can be ligated into this plasmid when used in conjunction with any 5 prime site (NotI-NcoI). To add this overhang the variable region must be PCR amplified to contain any of the following sites at its 3 prime end: BseRI BsgI BtsI or BsrDI. By using this system it allows antibody variable regions to PCR amplified and fused to any of our constant region plasmids without having to re-synthesise the entire antibody expression cassette each time.

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