PSF-CMV-HULAMBDA LC - HUMAN LAMBDA LIGHT CHAIN ANTIBODY VECTOR

An antibody expression plasmid designed for the production of human antibody lambda genes by fusing variable antibody fragments with the constant domain in this plasmid. This can be achieved because the contstant region coding sequence in this plasmid is positioned downstream of a BseRI restriction site which when cleaved will produce an overhang consisting of the first two nucleotides of the first codon of the constant region. This allows variable antibody fragments to be seamlessly inserted by placing a BseRI site in the opposing direction at the end of the variable fragment. We also provide plasmids for the expression of both rat and mouse antibodies as well as vectors for the expression of human IgG heavy chain and Kappa light chain (called pSF-CMV-HuIgG1 HC and pSF-CMV-HuKappa LC respectively)

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.

The plasmid encodes a constant region an antibody in the main multiple cloning site positioned so that it can be cleaved to produce an overhang that allows seamless fusion with a variable region from any antibody. This allows you to create full length 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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