Research On The High Quality Expression Of Aβ42 Peptide With The E. Coli Preferred Codon

Alzheimer’s disease(AD) is a neurodegenerative disease which mostly occurs in older age groups, with the symptoms of cognitive impairment and memory deficits of the central nervous system, the typical pathological characteristics are senile plaques (senile plaques, SP) and neurofibrillary tangles (neurofibrillary tangles, NFT) and primary neuronal degeneration and necrosis.β-amyloid (Aβ) deposition in the brain tissue may be the main cause of AD occurrence and development . Currently, the toxic research of Aβ’s effects on neurons in vitro and the research of interactions between Aβand the relating proteins on the pathways of signal transduction requires large amounts of recombinant Aβ. To date, most of the Aβ42 peptides are usually synthesized by chemical equipment, which is time-consuming, sophisticated and expensive,therefore,finding an economic and efficient method to obtain high level expression of recombinant Aβbecomes an urgent necessity.In this study, an E. coli expression system of Aβ42 was constructed by inserting the Aβ42 E. coli preferred codon in the plasmid pET28a, Aβ42 peptide was expressed without tag in the BL21 strain intracellularly. Based on the main characteristics of Aβ42 peptide, such as the small molecular weight of 4.5 kDa, low PI of 5.6, and its high amyloidogenic properties, denaturation and renaturation of inclusion bodies followed by ion exchange chromatography were performed for purification of Aβ42 peptide. Inclusion body extraction, dissolution and a variety of complex approaches for protein refolding, anti-dilution refolding were compared with each other to obtain the highest level of soluble protein. To induce the supernatant expression, different culturing conditions including adding different kinds of salt ions in the culture medium were performed, the results showed that Aβ42 peptide was abundantly secreted in the cell supernatant when culturing in the LB medium with 0.75mmol/L IPTG at 37℃for 3 hours, adding sodium acetate can effectively promote the expression of Aβ42 peptide, with the sodium acetate concentration range between 50mM / L to 200mM / L, the increase of the expression consists both the supernatant and pellet parts. This study also examines the different pH values for Aβ42 peptide induced expression, indicating that Aβ42 peptide expression is changed under the conditions of different pH values.