Biochemical and Immunological Characterization of the DNA Binding Protein (RBP-Jx) to Mouse Jx Recombination Signal Sequence

Abstract

We have investigated whether Jx recombination signal sequence (RS) binding protein (RBP-Jx) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-Jx protein purified by Jx-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-Jx protein, recombinant RBP-Jx was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-Jx produced in silkworm cells could bind Jx-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-Jx has the ligase activity, the RBP-Jx protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-Jx fusion protein which was synthesized in E. coli and unable to bind Jx-RS. Using the anti-RBP-Jx monoclonal antibody we have shown that the RBP-Jx protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-Jx protein is consistent with the hypothesis that the RBP-Jx protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].