Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein

The mammalian aspartic proteinases procathepsin D and pepsinogen form insol uble inclusion bodies when expressed in bacteria. They become soluble but n onnative when synthesized as fusions to the carboxy terminus of E. coli mal tose-binding protein (MBP). Since these nonnative states of the mio asparti c proteinases showed no tendency to form insoluble aggregates, their biophy sical properties were analyzed. The MBP portions were properly folded as sh own by binding to amylose, but the aspartic proteinase moieties failed to b ind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of t he fusions was resistant to proteolysis. The fusion between MBP and catheps in D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP-procathepsin D and MBP-pepsinogen revealed species with ve ry large and heterogeneous sedimentation values. Refolding of the fusions f rom 8 M urea generated proteins no larger than dimers. Refolded MBP-pepsino gen was proteolytically active, while only a few percent of renatured MBP-p rocathepsin D was obtained. The results suggest that MBP-aspartic proteinas e fusions can provide a source of soluble but nonnative folding states of t he mammalian polypeptides in the absence of aggregation.