Improved method for expression of Kunitz-type serine proteinase inhibitor domain of beta-amyloid protein precursor in Escherichia coli and characterization of disulfide bonds of the product.
Kunitz-type serine proteinase inhibitor (KPI) domain of Alzheimer's disease-related beta-amyloid protein precursor (APP) was expressed in Escherichia coli as a fusion protein with a truncated form of Staphylococcus protein A. The fusion protein was purified from the cell culture medium using an IgG Sepharose column. The KPI domain was separated from the protein A portion by cleavage with human alpha-thrombin at the engineered recognition sequence, followed by purification on IgG Sepharose and reversed-phase HPLC columns. The recombinant KPI domain strongly inhibited trypsin; the inhibition constant (Ki) for bovine trypsin was 2.5 x 10(-11) M, comparable to those of the secreted forms of APP with the KPI domain. The recombinant protein contained three intramolecular disulfide bonds, which were determined to be located between Cys-6 (C1) and Cys-56 (C6), Cys-15 (C2) and Cys-39 (C4), and Cys-31 (C3) and Cys-52 (C5) of the recombinant KPI domain, respectively. These positions are highly homologous to those of disulfide bonds in bovine pancreatic trypsin inhibitor. The trypsin-inhibitory activity of the recombinant protein was abolished by preincubation with 0.4 mM dithiothreitol under non-denaturing conditions. By this mild reduction, all the disulfide bonds were completely cleaved. These results clearly indicate that the disulfide bonds play an important role in the function of the KPI domain of APP.
Oyama
F
F
Division of Biomedical Polymer Science, Fujita Health University, Aichi.
Hashino
K
K
Oyama
R
R
Kato
I
I
Titani
K
K
D000544:Alzheimer Disease / Q000201:enzymology; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013917:Thrombin; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D016564:Amyloid beta-Protein Precursor; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D013917:Thrombin