Analysis Of Protease Involved In Maturation Of Protein PXO₀3968from Xanthomonas Oryzae Pv. Oryzae

Xanthomonas oryzae pv. Oryzae, a Gram-negative bacteria, causes rice bacterial disease. Studies have shown that PXO₀3968encodes a sectreted protein and it needs proteases to to produce a17peptide which highly conserved in blight bacteria. To search the related protease（s）, followi ng studies have been done. Based on FRET, a protease detector was built; extracellular proteins from Xanthomonas oryzae pv. Oryzae （PXO99） were preliminary separated and analyed; putative proteases involved in maturation of PXO₀3968were cloned, expressed and purified.Proteases, also called proteolytic enzymes. They are involved in a number of biological processes of organisms:location and activity regulation of proteins, protein-protein interactions, signal transductions of the intracellular signali ng molecule, and generation of new bioactive molecules. Protease is the cutting-edge research in biology.There are two common methods of biochemical analysis to detect protease activity:the homogeneous detection and separation-based detection. In the homogeneous detection, the signal transition detected from reaction mixture without further processi ng indicates substrate turnover.In separation-based detection, the product and substrate should separated from the reaction mixture after the enzymatic reaction is stopped and quantified to indicate the enzyme activity. In homogeneous detection, fluorescent labeling techniques are primarily used to detect protease activity. Currently there are four methods widely used:a. The change in fluorescence intensity of a fluorophore chemically quenched by amide linkage to the peptide.b. The change of fluorescence resonance energy transfer（FRET）.c. The change of fluorescence intensity by a non-fluorescent dye interaction with fluoropHore.d. The change in the intensity of fluorescence polarization.In separate detection, the substrate and the product could be separated by high performance liquid chromatography, and quantified by UV or mass spectrometry. With genomic DNA of PXO99, PXO₀3968was amplified and inserted between CFP and YPF with restriction enzymes.This prokaryotically epressed fusion protein forms inclusion bodies, and no FRET signal was detected even after denaturationa and renaturation. Insertion of predicted cleavage site sequence between CFP and YPF, yielding the prokaryotically protein have FRET signal, we refer this as the proteinase detector.To obtain a crude mixture of proteases, PXO99bacterial culture supernatant was concentrated by ultrafiltration. We found that fractions over30kDa can reduce FRET signal significantly. This indicated that there are one or several specific protease which can cleavage predicted cleavage site sequence in this fraction.After analysis with mass spectrometry, following proteases were YP001914183.1; YP₀01914215.1; YP001916133.1; YP₀01911256.1; YP001911714.1. Ultimately, only periplasmic protease（YP₀01914215.1） was successfully expressed and purified, and found that it can cleavage PXO03968between amino acid N and F by protein N-terminal sequencing.The work done here provided some preliminary data for further analysis the functions of PXO03968.