The Expression,Identification And Purification Of Recombinant 4-Hydroxyphenylpyruvate Dioxygenases From Human And Arabidopsis Thaliana

4-Hydroxyphenylpyruvate Dioxygenase(HPPD) is a class of a-keto acid-dependent non-heme iron(II) oxygenases which can be widely found in mammals, plants and most microbes. HPPD catalyzes oxygenation of 4-hydroxyyphenylpyruvate(HPP) to generate homogeritisate(HG). In plants, the biosynthesis of plastoquinones and tocopherols will be prevented once HPPD is inhibited, which leads to the decrease the formation of carotenoids, blocking of photosynthetic electron transfer chain and photooxidation of chloroplasts. These make plants bleach to death finally and HPPD is therefore selected as a target for herbicides.This thesis is mainly focused on expression, identification and purification of recombinant HPPD, including the following three aspects:1. DNA fragments of human HPPD were amplified by polymerase chain reactions(PCR). After being digested by enzymes, the correct DNA fragments were inserted into pET-28a expressive vectors by genetic recombination technology. Then the recombinant plasmids were transformed into Escherichia coli BL21 cells to overexpress the target enzyme, followed by identification with sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE), western blot and enzyme assays. The enzyme with high activity and high purity was abtained through affinity chromatography and Michaelis constant Km for the substrate was determined as 33 ?M. Therefore, the whole system of expression, identification and purification for recombinant human HPPD has been established.2. The construction of arabidopsis thaliana HPPD plasmids could be completed via DNA fragments of arabidopsis thaliana HPPD and pET-15b vectors in the same way as recombinant human HPPD plasmids. The recombinant plasmids were transformed into BL21 cells and the target enzyme was overexpressed and identified. This enzyme was purified through affinity chromatography and ion exchange chromatography, then enzyme characterization was performed and Michaelis constant Km for the substrate determined as 12.8 ?M.3. Purification of the two enzymes in a large scale provided support for protein crystallization. Meanwhile, the system established for enzymic activity assay with the coupled method provided a solid foundation for high-throughput screening of HPPD inhibitors at the level of enzymic activity in vitro.