Recombinant Production of B-Cell Receptor Fab Fragments from Chronic Lymphocytic Leukemia in Mammalian Cells for Structural and Biochemical Studies

Chronic lymphocytic leukemia (CLL) arises from the uncontrolled proliferation of neoplastic B cells that clonally express B-cell receptors (BcR) able to trigger the intracellular signaling cascade also in an exogenous antigen-independent manner. This cell-autonomous signaling has been shown to involve the establishment of clone-specific intermolecular BcR–BcR interactions that initiate Ca2+ ion influx and target gene transcription. CLL BcRs are characterized by a variability in gene usage and paratope structure that parallels the heterogeneous disease progression, but homologous BcRs with stereotyped HCDR3 isolated from groups of patients (subsets) are associated with a more homogeneous clinical outcome. To fully understand the molecular mechanism underlying the activation of the individual neoplastic CLL B cells, an efficient workflow for the recombinant production of the antigen-binding fragment from CLL BcRs is required. Here, we describe a protocol for the expression and purification of BcR-derived Fabs in mammalian cells that can be used for studies of self-association in solution as well as experimental structure determination using X-ray crystallography.