Cloning, High Prokaryotic Expression And Purification Of Human Beta Amyloid Protein

Several biochemical and molecular studies point to mechanisms by which Abeta is generated and aggregated to trigger the neurodegeneration that may cause Alzheimer's disease (AD). In diagnostic and therapeutic field of AD, Abeta would play a more significant role in near future. Based on technology of PCR and DNA recombination, the prokaryotic expression plasmid was constructed in this paper. Then the optimization of the fermentation conditions with shaking-flask for GST-Abeta in E. coli DH5αwas also studied. Finally, the separation and purification of GST-Abeta were investigated with affinity chromatography.The results are as follows:1. Construction of the prokaryotic expression plasmidPairs of primers were designed according to human Abeta gene published in Genbank. Overlapping-PCR method and synthesized oligo-nucleotide primers were used to amplify the DNA sequence of human Abeta. Then the fragment was inserted into the TOPO-TA2.1 vectors and transformed into E. coli DH5α. After confirmed correctness of the DNA by analysis of PCR, digestion, and sequence, they were subcloned into pGEX-6p-1 and then transferred into DH5α. SDS-PAGE and Western blot analysis confirmed that the recombinant fusion protein of about 29KD was the target protein.2. Optimization of the fermentation conditions with shaking-flaskThe effects of inducing temperature and time on the growth of bacteria and expression of GST-Abeta were investigated. Also the process conditions, such as rotational speed and inoculum volume , were optimized. The results showed that it was suitable for inducing cultivation at 30℃for 4 hours when the growth of bacteria was in the initial logarithm growth phase, and...