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Expression, Purification and Characterisation of Full-Length B doi:10.1016/S0022-2836(02)00424-2 available online at http://www.idealibrary.com on w J. Mol. Biol. (2002) 320, 201–213 Expression, Purification and Characterisation of Full-length Histidine Protein Kinase RegB from Rhodobacter sphaeroides Christopher A. Potter1, Alison Ward1, Cedric Laguri2 Michael P. Williamson2, Peter J.F. Henderson1 and Mary K. Phillips-Jones1* 1Division of Microbiology The global redox switch between aerobic and anaerobic growth in School of Biochemistry and Rhodobacter sphaeroides is controlled by the RegA/RegB two-component Molecular Biology, University system, in which RegB is the integral membrane histidine protein kinase, of Leeds, Leeds LS2 9JT, UK and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no 2Department of Molecular reported examples to date of heterologous expression of full-length RegB Biology and Biotechnology or any histidine protein kinases. Here, we report the amplified expression University of Sheffield of full-length functional His-tagged RegB in Escherichia coli, its purifi- Sheffield S10 2TN, UK cation, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half- life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli ) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB. q 2002 Elsevier Science Ltd. All rights reserved Keywords: Rhodobacter sphaeroides; RegB; membrane receptor; Ni2þ affinity *Corresponding author purification; phosphorylation kinetics Introduction in oxygen-responsive regulation of nitrogen fix- ation genes.4 In Rhodobacter, RegB is the mem- The RegBA two-component system (also known brane-located histidine protein kinase (HPK) as PrrBA) serves as a major transcriptional regula- component of the system, sensing changes in tor of gene expression in several photosynthetic 1–6 redox conditions. Upon anaerobiosis, RegB and nitrogen-fixing bacteria. It has been studied becomes autophosphorylated in an ATP-depen- most intensively in Rhodobacter sphaeroides and dent reaction; RegB , P then transfers the phos- R. capsulatus, in which RegBA is a globally acting, 7 phoryl signal to Asp63 of the partner response redox-responsive system, and in nitrogen-fixing regulator RegA.8,9 Once phosphorylated, Bradyrhizobium japonicum, in which it is involved RegA , P then positively regulates photosynthesis gene expression ( puc, puf and puhA ),1–3 as well as expression of genes involved in carbon dioxide Present address: A. Ward, Astex Technology Ltd, 250 10,11 fixation (cbbI and cbbII operons), nitrogen Cambridge Science Park, Cambridge CB4 0WE, UK. fixation (nifA2 ),7,12 electron transport functions Abbreviations used: HPK, histidine protein kinase; b D ( petABC, cycA, cycY ) and respiratory terminal elec- TMR, transmembrane region; DDM, dodecyl- - - 3,13,14 maltoside. tron functions (cydAB, ccoNOPQ, dorCBA ). E-mail address of the corresponding author: RegA , P also negatively regulates hydrogenase [email protected] expression (hupSLC ).12 Here, we retain the name 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved 202 Activities of Full-length RegB Figure 1. SDS-PAGE and Western analysis of mixed membrane proteins from IPTG-induced E. coli NM554 cells carrying pTTQregB (full-length regB gene) and/or pTTQEP6 (truncated regB gene). (a) SDS-PAGE of mixed membranes of E. coli NM554 (pTTQregB ) uninduced or induced with 1 mM IPTG and resolved using 15% polyacrylamide gels and visualised by staining with Coomassie brilliant blue. Lane 1, uninduced mixed membranes; lane 2, IPTG-induced mixed membranes; lane 3, molecular mass markers. (b) Western analysis of mixed membranes from E. coli (pTTQregB ) and E. coli (pTTQEP6 ). Lane 1, molecular mass markers (no His-tag). Lane 2, E. coli NM554 (pTTQEP6 )(1mg of mixed membrane protein). Lane 3, E. coli NM554 (pTTQregB )(1mg of mixed membrane protein). Lane 4, E. coli (pTTQEP6 ) (5 mg of mixed membrane protein). Lane 5, E. coli NM554 (pTTQregB )(5mg of mixed membrane protein). Reg (rather than Prr) for the R. sphaeroides system, redox sensing.19 – 21 ArcB, like RegB, senses redox for consistency with the homologues identified in signals via the state of components of electron other bacterial species, including R. capsulatus,in transport; oxidised forms of quinone electron which the Reg system was first described.1 carriers serve as negative signals that inhibit auto- RegB senses redox through changes in the phosphorylation of ArcB during aerobiosis.19 In volume of electron flow through the cbb3-type cyto- the case of ArcB, the TMRs serve as membrane chrome c oxidase of the respiratory electron trans- anchors, playing no detectable role in sensing or port chain.15 The precise mechanism is not signal transduction.19 However, this does not elucidated, but may be mediated by the SenC appear to be the case for RegB. Recent in vivo muta- protein.16,17 In vivo studies suggest that under genesis studies of the transmembrane domain of aerobic conditions, the volume of electron flow R. sphaeroides RegB protein revealed the import- through cbb3 oxidase is sufficiently high to repress ance of a central portion of this domain, particu- the default kinase-positive mode of RegB, resulting larly the short second periplasmic loop and in repression of the autophosphorylation of RegB.9 membrane-spanning a-helices 3 and 4, for sensing Anaerobic conditions relieve this repression and and signal transduction.9 Thus, in future studies result in autophosphorylation of RegB and sub- of the signal-sensing and transduction mechanism, sequent signal transfer to RegA.9 full-length versions of RegB that include the trans- R. sphaeroides RegB (462 amino acid residues) membrane regions will be required. However, possesses two domains, an N-terminal trans- previous attempts to express full-length RegB and membrane domain (residues 1–182) predicted to its homologues8,22 – 24 have consistently failed to comprise six membrane-spanning regions, and a obtain this protein in a soluble, folded and func- cytosolic C-terminal histidine kinase domain tionally active form; indeed, of all the membrane (residues 183–462) for autophosphorylation, phos- HPKs reported to date, only Escherichia coli phatase and phosphotransfer reactions.18 It is an KdpD25 and NarX26,27 have been expressed success- atypical HPK because it appears to lack any poten- fully as enriched proteins in E. coli and utilised in tial signal-sensing periplasmic domains between functional assays. Of these two proteins, only the transmembrane regions (TMRs). This implies NarX26 retains functional activity after purification either that RegB signal sensing occurs within the from membranes. KdpD regains activity only after TMRs themselves, or that signals are sensed else- reconstitution into membrane vesicles.25 Other where within the soluble domain, with TMRs studies, including those of RegB, have used trun- merely serving as anchors to keep the protein in cated versions that lack the transmembrane close contact with the source of the signal. This domains.8,23,24 lack of significant periplasmic domains is charac- In order to address this problem, we utilised teristic also of ArcB, another HPK involved in plasmid pTTQ18His, a membrane protein Activities of Full-length RegB 203 expression plasmid that has been used for the successful overexpression of 16 membrane proteins,28 – 30 to amplify expression, and sub- sequently isolate and purify, intact RegB. This is the first successful overexpression of an HPK in an heterologous E. coli host. The overexpressed protein is functional in E. coli inner membranes, as shown by its autophosphorylation activity and its ability to be dephosphorylated by RegA. Impor- tantly, it is functional following purification, since autophosphorylation, phosphotransfer and RegA- dephosphorylation activities were all demon- strable. Our kinetic data obtained for this intact protein reveal important differences compared with the truncated version of soluble RegB from R. sphaeroides,24 demonstrating that the trans- membrane region has important regulatory activity. Figure 2. Autophosphorylation of RegB in E. coli inner membranes under aerobic conditions in the presence of Results [g-33P]ATP. Each reaction employed 20 mg of purified inner membranes obtained from IPTG-induced E. coli Expression of RegB NM554 (pTTQEP6 ) (lanes 2 and 4) or NM554 (pTTQregB ) (lanes 3 and 5). Reactions (20 ml final The full-length regB gene was cloned into the volumes) were incubated at 24 8C with 50 mM ATP, pTTQ18His plasmid, to produce plasmid 5 mCi of [g-33P]ATP and 1 mM DTT for ten minutes pTTQregB coding for the C-terminally His-tagged prior to the addition of 3 pmol of RegA (lanes 4 and 5). protein RegB-His6 (referred to as RegB). Following RegA was added to a reaction containing no inner introduction
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