Expression, purification and the biological activites of C-terminal fragment CTFβ

Objective Proteolysis of the C-terminal fragment (CTFβ) of the amyloid precursor protein (APP) generates the Aβ peptides associated with Alzheimer' s disease (AD).The metabolism of CTFβ may play key roles in early stage of AD before Aβ generation.The aim of this study was to express,identify and purify the CTFβ,so as to provide evidence for its application in the development of AD detection system.Methods APP gene was used as the template,and the gene of CTFβ was cloned to pMD18-T vector through PCR.After sequencing,the CTFβ gene was cloned into the expression vector pET-30a(+) to construct the recombinant expression plasmid pET30a-CTFβ.The expression plasmid was transformed into Escherichia coli BL21 and the expression of CTFβ was induced by Isopropyl-β-D-thiogalactoside (IPTG).The effect of expression was confirmed by Western blottng.The recombinant protein was purified by using Ni-NTA affinity chromatography column,and the immunoreactivity of recombinant protein was detected by Western blotting and indirect ELISA.Results Western Blot results showed that recombinant protein was solubly expressed in E.coli and its molecular weight was about 10 × 103 to 25 × 103,and the size of fusion protein was consistent with prediction.In addition,the data of Western blotting showed that there were still some thick bands above the position of 80 × 103.Furthermore,the immunoblotting demonstrated that the 10× 103 to 25 × 103 of monomer of fusion protein was recognized by anti-histidine (his) tag and anti-Aβ (17-24) (4G8) antibody,while the high molecular aggregates were above 80 × 103 which were detected respectivly by anti-his,anti-Aβ antibody NU1,NU4 and A8.However,our data suggested that the bands above 80 × 103 were poorly recognized by fibril specific antibody NU6.These results demonstrated that the purified recombinant protein showed a specific immunoreactivity.Finally,indirect ELISA showed that the optimal concentration of CTFβ to coat the ELISA plate was 1 ng/well when it was used to detect Aβ antibody.Conclusion In this study,one of the aggregate-prone fragment of APP,CTFβ,was successfully expressed,purified,and identified,which would provide experimental clue to futher application in biochemical diagnosis of AD. Key words: Alzheimer disease;  Protein conformation;  Prokaryotic expression