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| Construction of Pichia pastoris Expression Vector of Codon Optimized αB-crystallin Gene and Expression Optimization |
| FENG Xue, GAO Xiang, NIU Chun-qing, LIU Yan |
| College of Life Science, Southwestern University, Chongqing 400715, China |
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Abstract αB-crystallin is a member of the small heat shock protein family,which has the molecular chaperones activity. The expression of αB-cr ystallin increases obviously in astrocytes in the brains of Alzheimer's disease (AD) patients, and colocalizes with β-amyloid (Aβ) protein deposition densely. In order to study the mechanism of extracellular αB-crystallin in Alzheimer's disease at molecular level, need to obtain a large amount of active αB-crystallin. According to the Pichia pastoris codon preference, the codons of αB-crystallin gene and added six histidine residues at the 3' terminal of the gene. Then the optimized gene is claned into pPIC9K and transferred the vector into Pichia pastoris GS115 is optimized through the electroporation method. The best expression conditions are as follows:the transferring proportion of BMGY and BMMY is 2:1; the expression time is 72h and adding the methanol to the final concentration to 0.5%. There are three protein fragments by Western blotting, and the molecular weights of them are:20.0kDa, 18.6kDa and 13.1kDa. The recombination protein was partially degraded and obtained the purified 20kDa fragment, which has molecular chaperones activity using insulin reduction method. The result may make a base line for further studing protection mechanism of exogenous αB-crystallin to astrocytes in Alzheimer's disease.
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Received: 04 January 2017
Published: 25 July 2017
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