Expression and characterization of a plant cis-prenyltransferase LLA66 in Saccharomyces cerevisiae
| Autor: | Jyun-Yu Yao, 姚俊宇 |
|---|---|
| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths by consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). According to the stereochemistry of the double bonds formed by IPP condensation, these prenyltransferase are classified as cis- and trans-types. Cis-prenyltransferases synthesize longer products as lipid carriers to mediate biosynthesis of peptidoglycan in bacteria or glycoproteins in eukaryotes. However, the functions of cis-prenyltransferases in plants are less understood because there are several homologous isoforms in different compartments. The cis-prenyltransferase gene LLA66 from Lilium longiflorum anther is the first prenyltransferase identified in the tapetum and microspores. As reported here, we have produced the recombinant LLA66 in Saccharomyces cerevisiae, performed in-vitro characterization of the enzyme and found it is a unique medium-chain synthase, and was resolved C45 isoprenoids in thin layer chromatography (TLC) and HPLC. After purification of the hexa-His-tagged LLA66 using Ni-NTA affinity chromatography, we also identified enzyme activities to convert IPP into farnesol in the purified protein mixture. By further analysis, we thought that a protein mixture of IPP:DMAPP isomerase, FPP synthase and alkaline phosphatase was co-purified with LLA66. We will further characterize the protein complex. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|