title: One-step expression and tyrosine O-sulfonation of Ax21 in Escherichia coli.
abstract: Ax21 (activator of Xa21-mediated immunity), a pathogen-associated molecular pattern secreted by Xanthomonas oryzae pv. oryzae, can be perceived by a membrane-located pattern recognition receptor Xa21 and triggered immune responses in rice. An Ax21-derived peptide (17-amino acid) containing a sulfated tyrosine-22 (axY(S)22) is sufficient for Ax21 activity. Here, we expressed Ax21 and O-sulfated its tyrosine-22 through coexpressing a putative tyrosine sulfotransferase, raxST, and two other genes involved in the synthesis of 3'-phosphoadenosine 5'-phosphosulfate in Escherichia coli BL21 (DE3). The sulfated Ax21 fused with a histidine tag in its N-terminus was extracted and bound onto a Ni-NTA agarose and then cleaved with Factor Xa and CNBr in turn. Δax21Y(S)22, a 36-amino acid peptide covering axY(S)22 in the lysate supernatant, was finally yielded after ultrafiltration. The purified peptide was further verified by Tricine-SDS-PAGE and isoelectrofocusing electrophoresis. Lesion length analysis, reactive oxygen species production, and mitogen-activated protein kinase (MAPK) activation of rice leaves inoculated with Δax21Y(S)22 confirmed the activity of the sulfated peptide. Overall, this study successfully established an efficient system for expression and purification of a sulfated peptide. In addition, the sulfotransferase activity of RaxST was confirmed for the first time.
authors.affiliation: School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, China. biohou@sdjzu.edu.cn
authors.forename: Hou
authors.initials: H
authors.lastname: Shuguo
authors.forename: Zhang
authors.initials: Z
authors.lastname: Wei
authors.forename: Zhang
authors.initials: Z
authors.lastname: Chao
authors.forename: Wu
authors.initials: W
authors.lastname: Daoji
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D012275:Oryza / Q000187:drug effects / Q000276:immunology; D010446:Peptide Fragments / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects / Q000276:immunology; D013451:Sulfonic Acids / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014461:Ultracentrifugation; D014974:Xanthomonas / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D013451:Sulfonic Acids; D014443:Tyrosine