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.

TheXanthomonasAx21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

“…Some of the retracted results have been reproduced in other laboratories (Shuguo et al, 2012; Wang et al, 2013). For example, Shuguo et al (2012) showed that a sulfated, 36-aa peptide, derived from the N-terminus of Ax21, can induce various defense responses in XA21-expressing rice such as accumulation of reactive oxygen species, mitogen-activated protein kinase activation and in planta XA21-mediated immunity.…”

“…For example, Shuguo et al (2012) showed that a sulfated, 36-aa peptide, derived from the N-terminus of Ax21, can induce various defense responses in XA21-expressing rice such as accumulation of reactive oxygen species, mitogen-activated protein kinase activation and in planta XA21-mediated immunity. Wang et al (2013) showed that ax21 deletion mutants in both Xoo (strain PXO99) and X. oryzae pv.…”

MAMP (microbe-associated molecular pattern) triggered immunity in plants

“…raxA and raxB are arranged in a putative operon (called raxSTAB ) together with the gene raxST , which is not a part of the TOSS (da Silva et al, 2004). Instead raxST is a sulfotransferase (Shuguo et al, 2012), which catalyze the transfer of sulfate from PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a tyrosine residue. Sulfation of secreted peptides is often important for their biological function.…”

Expression, Purification and Characterization of Recombinant Histidine-tagged L-asparaginase II

“…DNA nucleotides coding for the polyhistidine affinity tag can also be created from synthetic oligonucleotides and cloned into an appropriate location in the desired plasmid. Alternatively, there are a wide variety of commercially available expression vectors for expression of polyhistidine-tagged recombinant proteins in different expression systems (Rabbani et al, 2010 andShuguo et al, 2012).…”