Expression And Purification Of Lipoxygenase And Establishment Of The Inhibitor Screening Assay

Aim: To express and purification5-LOX and15-LOX protein in Escherichia coli （E. coli）anddetermine the bioactivity assay. It will provide an experimental basis for the inhibitor screeningof5-LOX and15-LOX.Methods: The full length of human5-Lipoxygenase (h5-LOX) and mouth15-Lipoxygenase(m15-LOX) cDNA obtained through PCR amplification were inserted into the vector pET15band were transformed into Escherichia coli (E. coli) Rosetta-gami strain for expression. Then theexpression conditions were optimized to obtain the high purified product, contained theconcentration of isopropyl-β-D-thiogalactopyranoside (IPTG), the culture temperature and time,The expression product were identified by SDS-PAGE. After optimized, the recombinant proteinwas refolded in vitro. HPLC was employed to analyze the bioactivity of h5-LOX and m15-LOXand screen the inhibitors.Results: Through PCR amplification obtain the h5-LOX gene at2025bp and the m15-LOX geneat1992bp. Nucleotide sequencing proved that recombinant plasmid pET-15b-h5-LOX andpET-15b-m15-LOX were constructed correctly. After induced, expected molecular mass of80.8kDa for h5-LOX and78.0kDa for m15-LOX were detected by SDS-PAGE mainly in insolubleform. The concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) as an inducer, thetemperature and culture time after induction were adjusted to0.1mmol/L,25℃,24h for optimalexpression. Subsequently, the optimal purification condition (1.5mol/L urea washing solution)was determined by comparing the washing effects of various concentrations of urea solutions,and the percent of h5-LOX in total protein was raised from12.6%to69.2%before purification,compared to m15-LOX from5.4%to64.2%. Then inclusion bodies were dissolved in8mol/Lurea and refolded by the method of dialysis renaturation. Furthermore, the inhibition ofdicaffeoylquinic acids on h5-LOX and m15-LOX was analyzed by HPLC. The IC50of BR1, BR2,BR3and BR4on5-LOX was9.30.20μmol/L、27.10.88μmol/L、25.92.88μmol/L、3.10.54μmol/L, respectively, and on15-LOX was6.04.12μmol/L、16.72.35μmol/L、6.90.96μmol/L、7.54.71μmol/L, respectively. The results showed that these dicaffeoylquinic acids (DCQs) could significantly inhibit the activity of h5-LOX and15-LOX in the metabolism ofarachidonic acid.Conclusion: Proteins were successfully expressed in Escherichia coli Rosetta-gami strain andthe condition of expression and purification were studied. Furthermore, in vitro bioactivity assay,we determined four dicaffeoylquinic acids inhibition effects on h5-LOX and m15-LOX.