Protein Expression of E.coli pET23a-FPO for HbA1c detection

• Main Authors: Chen-Wen Liao, 廖貞玟
• Other Authors: Cheng-Kang Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/54470937849645035493

碩士 === 國立臺灣科技大學 === 化學工程系 === 101 === HbA1c (Hemoglobin A1c) is the major glycated hemoglobin in human blood, the amount of the HbA1c reflects the average blood glucose concentration of the past few months. HbA1c is recognized as an important diagnostic indicator of diabetes mellitus. HbA1c is a hemoglobin molecule in which the N-terminal valine residue of its β－subunit has been modified by blood glucose. In this study, the enzymatic method was developed to detect HbA1c concentration in blood. It consists three steps: (1) proteolysis of the HbA1c β－subunits to release fructosyl valine (FV) (2) oxidation of the released FV by FPO (fructosyl peptide oxidase) to produce H2O2 (3) detection of the produced H2O2 FPO was expressed by a genetic engineered E.coli pET23a－FPO in this work. The recombinant E.coli strain cultured at 26℃ was found out can produce more FPO activity than at 37℃. The specific activity of purified FPO protein is 26.01 U/mg after IMAC purification. It is about 5 fold higher than that of commercially available FAO. The FPO protein can well maintain its activity in the range of 4℃~ 40℃ and pH 5~10. In addition, the FV detection sensitivity using the dye of ODA and DA-64 has been investigated. The results showed that the sensitivity of the DA-64 was 6.3 fold higher than that of ODA. FV concentration as low as 0.02Mm could be detected by using this enzymatic colorimetric method.