scFv/Fab Construction

Creative Biolabs has established a solid platform for scFv and Fab construction. There are three routes to have a scFv or Fab targets a specific antigen. The first approach is to generate a mouse hybridoma clone, then convert full IgG (or IgM) into a scFv or Fab; the second approach is to create an immunized phage display scFv or Fab mouse library, then use the antigen to screen the library; the third method is to use the antigen to screen a premade scFv or Fab antibody phage display library (usually a human one) to get scFv or Fab clones directly. We are also able to convert chicken IgY into scFv or Fab fragments.

Fab fragments differ from scFv's as they contain both variable domains and constant regions, thus their construction and ultimate expression is somewhat more complex. Creative Biolabs is experts with many years of experience in antibody production. The same heavy and light variable chains used for scFv construction can be used in the construction of Fab.

If a monoclonal antibody is available, the Fab fragment can be created enzymatically. papain is a cysteine protease enzyme that has the endopeptidase activity to cleave immunoglobulin G molecules in the hinge reason. This cleavage results in the generation of three ~50kDa fragments, two Fab domains and a Fc domain. The papain-digested antibody is unable to promote agglutination, precipitation, opsonization, and lysis. Immobilized papain offers the advantage of generating Fab and Fc fragments without the need to remove the papain enzyme after digestion.

Expression strategies:

- Secreted expression in periplasm of E. coli.

- Soluble expression in cytoplasm of E. coli.

- Insoluble expression as inclusion bodies.

- SUMO Fusion Platform.

Purification strategies:

- Affinity tag: Hexahistidine (His6) tag.

- Molecular format: insertion of SUMO between His6 tag and protein of interest (POI).

- Purification: IMAC (Immobilized Metal Affinity Chromatography).

- Tag removal after purification: cleavage at the C terminus of SUMO with SUMO protease.