General approach for isolation of immunoglobulins fragments with the core hinge

The original limited proteolysis approach was proposed for large (multidomain) proteins with post-translational modifications for obtaining of their novel fragments. It was realized for human immunoglobulins representing two subclasses IgG2 and IgG3. This approach was based on two techniques: masking of protein regions which are normally susceptible to proteolytic enzymes and increasing the possibility of proteolysis for sites which are not ordinarily accessible to these enzymes. The masking of immunoglobulin part which is sensitive to proteolysis was performed by Fab fragments (Fv subfragments) of antibodies and the increase of lability of stable regions was realized by pH change and mild reduction of disulfide bonds.