The progressive cerebral deposition of a 40-42 residues amyloid beta-peptide (A beta) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that A beta(1-40) is cleaved by Escherichia coli pitrilysin, a homolog e of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of A beta by pitrilysin, an overproduction system of A beta(1-40) as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an A beta(1-40) derivative, A beta(1-40)*, in which Lys(16) and Lys(28) of A beta(1-40) are simultaneously replaced by Ala, is attached to the C-terminus of E coli RNase HI and A beta(1-40)* is separated from RNase HI upon cleavage with lysyl endopeptidase. The fusion protein was overproduced in E. coli in inclusion bodies, solubilized and purified in the presence of guanidine hydrochloride, and cleaved by lysyl endopeptidase. A beta(1-40)* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that A beta(1-40)* is conformationally similar to A beta(1-40). However, the thioflavin T binding assay suggests that A beta(1-40)* is more amyloidogenic than A beta(1-40). Nevertheless, A beta(1-40)* was cleaved by pitrilysin at the site identical to that in A beta(1-40). (c) 2005 Elsevier B.V. All rights reserved.
