The objective of this project is to employ a bacterial high-level expression system to express recombinant ribonuclease of frog (Rana Pipiens) origin (rpRNase), either alone, or as a fusion protein with a humanized single chain antibody specific for B-cell lymphoma (rpRNase-hLL2scFv). Attempts to express rpRNase as recombinant protein were not successful because of its cytosolic toxicity in mammalian cells and its necessary requirement for an N-terminal pyroglutamyl residue to remain enzymatically active. The proposal suggests the production of rpRNase or rpRNase-hLL2scFv in E. coli as inclusion bodies containing an N-terminal Methionine. Strategies are developed to solubilize and renature the recombinant proteins which are post-translationally modified to resume a bioactive structure. The biological activities of the recombinant proteins in terms of RNase activity, inhibition or protein synthesis in cell-free system or in tissue culture will be studied. For the immunotoxin fusion protein, a cleavable peptide linker is incorporated to facilitate rpRNase release upon internalization. The bioactive recombinant rpRNase will be chemically conjugated to hLL2, and its anti-tumor effect, both in vitro and in vivo (in tumor-bearing mice), will be compared with that of rpRNase-hLL2scFv.
The development of an economic and efficient strategy to produce a bioactive recombinant rpRNase either alone or as fusion protein to a B-cell lymphoma specific single chain antibody. The recombinant rpRNase can be used as an anti-tumor agent for sensitize tumors or as immunotoxin conjugated to antibodies of different tumor specificities. The fusion protein can be used for the treatment of B-cell lymphoma.
