Purification of soluble and active RaxH, a transmembrane histidine protein kinase from Xanthomonas oryzae pv. oryzae required for AvrXa21 activity

Avital Stolov, Angel Valverde, Pamela Ronald, Saul Burdman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The RaxHR two-component regulatory system (TCS) of the rice pathogen Xanthomonas oryzae pv. oryzae is required for AvrXa21 activity. RaxH is a typical transmembrane histidine protein kinase (HK), whereas RaxR is its concomitant response regulator (RR). Here, we report the isolation of soluble, active amounts of recombinant His-tagged full-length RaxH and RaxR following growth of Escherichia coli over-expressing strains in the presence of sorbitol and glycine betaine. Full-length His-RaxH showed similar autophosphorylation activities to that of a truncated version of the protein (His-t-RaxH), lacking the N-terminal transmembrane region. Transphosphorylation assays revealed that only full-length RaxH was able to induce phosphorylation of His-RaxR, indicating that the N-terminal region of RaxH may be required for transphosphorylation of RaxR. Using site-directed mutagenesis we also demonstrated that residues histidine 222 in RaxH and aspartate 51 in RaxR are essential for phosphorylation activities of these proteins. Utilization of compatible solutes may be widely applied for purification of soluble, active recombinant transmembrane proteins, and in particular for purification of transmembrane HKs.

Original languageAmerican English
Pages (from-to)93-101
Number of pages9
JournalMolecular Plant Pathology
Volume8
Issue number1
DOIs
StatePublished - Jan 2007

Fingerprint

Dive into the research topics of 'Purification of soluble and active RaxH, a transmembrane histidine protein kinase from Xanthomonas oryzae pv. oryzae required for AvrXa21 activity'. Together they form a unique fingerprint.

Cite this