LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF RECOMBINANT FIBROBLAST GROWTH FACTOR-SAPORIN MITOTOXIN

In order to produce sufficient quantities of fibroblast growth factor- saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large- scale expression, purification, and characterization of the recombinan t molecule, The fusion gene encoding rFGF-2-SAP was cloned into the in ducible pET 11a expression vector and transformed into Escherichia col i strain BL21 (DE3). The transformants were grown using a fed-batch fe rmentation until the A(600) reached 85. At this stage, induction of th e expression of the fusion protein led to the production of similar to 2.2 mg/liter per A(600) unit. The soluble mitotoxin was purified to h omogeneity from cell lysates via expanded bed adsorption chromatograph y followed by cation-exchange, heparin-affinity, and size-exclusion ch romatography. Purified rFGF-2-SAP contained less than 0.5 EU/mg of end otoxin, as determined by gel clot analyses. The highly purified rFGF-2 -SAP retained the toxin's ability to inhibit protein synthesis as meas ured in a cell-free system and was cytotoxic to a number of normal and neoplastic cell lines bearing FGF receptors. Binding studies establis h that the fusion protein exerts its effects via the FGF high-affinity receptor. (C) 1996 Academic Press, Inc.