标题：One-step expression and tyrosine O-sulfonation of Ax21 in Escherichia coli
作者：Shuguo, H.;Wei, Z.;Chao, Z.;Daoji, W.
作者机构：[Shuguo, H] School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, China;[ Wei, Z] School of Life Science, Shandong Un 更多
通讯作者地址：[Hou, SG]Shandong Jianzhu Univ, Sch Municipal & Environm Engn, Jinan, Peoples R China.
来源：Applied biochemistry and biotechnology, Part A. enzyme engineering and biotechnology
关键词：Escherichia coli;PAMP;Plant immunity;Tyrosine O-sulfation;Xanthomonas oryzae pv. oryzae
摘要：Ax21 (activator of Xa21-mediated immunity), a pathogen-associated molecular pattern secreted by Xanthomonas oryzae pv. oryzae, can be perceived by a membranelocated pattern recognition receptor Xa21 and triggered immune responses in rice. An Ax21-derived peptide (17-amino acid) containing a sulfated tyrosine-22 (axY ~S22) 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 ~S22, a 36-amino acid peptide covering axY ~S22 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 ~S22 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.