Amyloidogenecity and pitrilysin sensitivity of a lysine-free derivative of amyloid β-peptide cleaved from a recombinant fusion protein

The progressive cerebral deposition of a 40–42 residues amyloid β-peptide (Aβ) is regarded as a major factor in the onset of the Alzheimer's disease. It has recently been shown that Aβ1–40 is cleaved by Escherichia coli pitrilysin, a homologue of insulysin, at a specific site. To facilitate the studies on a recognition mechanism of Aβ by pitrilysin, an overproduction system of Aβ1–40 as a fusion protein with E. coli RNase HI was constructed. This fusion protein was designed such that an Aβ1–40 derivative, Aβ1–40*, in which Lys16 and Lys28 of Aβ1–40 are simultaneously replaced by Ala, is attached to the C-terminus of E. coli RNase HI and Aβ1–40* 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. Aβ1–40* was purified from the resultant peptide fragments by reverse-phase HPLC. Measurement of the far-UV CD spectra suggests that Aβ1–40* is conformationally similar to Aβ1–40. However, the thioflavin T binding assay suggests that Aβ1–40* is more amyloidogenic than Aβ1–40. Nevertheless, Aβ1–40* was cleaved by pitrilysin at the site identical to that in Aβ1–40.