Prokaryotic Expression, Purification And Identification Of Human Recombinant β-amyloid Protein In E.coli

In recent years it has become evident that theβ-amyloid peptide component of senile plaques may be the key molecule in the pathology of Alzheimer's disease. In order to do the futher study of theβ-amyloid peptide, we developed a pGEX-4T-1-Aβ₄₂ recombinant vector by GST fusion system in E.coli. It confirmed by purification, identification and detection of its biological activity after digest.RT-PCR technology was employed to amplify the Aβ₄₂ gene with a pair of primers designed according to the nucleotides sequence of Aβ₄₂ gene pressed in GeneBank and the template DNA reversed by total mRNA extracted from SH-SY5Y. The gene and the plasmid pGEX-4T-1 were digested with Salâ… and BamHâ… respectively, and the gene was directionly cloned into the pGEX-4T-1. The recombinant vector pGEX-4T-1-Aβ₄₂ was characterized by restriction enzymes digestion and sequencing identification.The result demonstrated that the nucleotides sequence of cloned Aβ₄₂ gene was in correspondence with Aβ₄₂ cDNA pressed in GenBank accession.To improve the expression ofβ-amyloid peptide in Escherichia coli, various factors on the protein expression were studied, which included concentration of inductor IPTG, culture temperature, inducing time and sonicate condition. The results indicated that the soluble expression level of GST-Aβ₄₂ fusion protein could be promoted,when cells were cultured for 4 h at 37°C, and then shifted to 25°C for 2 h to induced the expression of GST-Aβ₄₂ with 1 mmol/L IPTG as final concentration. The GST-Aβ₄₂ fusion protein was purified by Glutathione Sepharose 4B affinity chromatography. SDS-PAGE analysis showed that the molecular mass of GST- Aβ₄₂ fusion protein was about 32 kDa as expected, and Western blot analysis indicated that it could specifically bind to both of the Aβ₄₂ antibody and GST antibody.The fusion protein GST-Aβ₄₂, expressed in BL21 (DE3) strain, was purified with Glutathione Sepharose 4B affinity chromatography followed by thrombin cleavage. The digested product was further purified with additional Glutathione Sepharose 4B affinity chromatography and Benzamidine affinity chromatography step. After enzymatic cleavage of GST tail, the Aβ₄₂ moiety was indicated a molecular weight corresponding to the expected 4.5 kDa by the analysis of Tricine-SDS-PAGE and Western blot. Moreover, the fibrillar recombinant Aβ₄₂ showed great biologically aggregation and toxicity property use cells multiplication, Hoechst 33342-PI and Thioflavin-T fluorescence assay.These results suggest that expression of human recombinantβ-amyloid protein in E.coli will be useful method in obtaining a large quantity of Aβ₄₂ peptide for further physiological and biochemical studies for Alzheimer's Disease.