ABSTRACT
Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography. Changes in HPLC retention times, chemical assays for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density for the phosphonomethyl group at Cys54, consistent with a modification at that position. Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY. Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with a dissociation constant of â¼29 µM, at least sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.
Subject(s)
Bacterial Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins/chemistry , Organophosphonates/chemistry , Peptides/chemistry , Thermotoga maritima/chemistry , Amino Acid Sequence , Aspartic Acid/analogs & derivatives , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Chemotaxis/physiology , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Flagella/chemistry , Flagella/metabolism , Gene Expression , Kinetics , Methyl-Accepting Chemotaxis Proteins/genetics , Methyl-Accepting Chemotaxis Proteins/metabolism , Models, Molecular , Organophosphonates/metabolism , Peptides/genetics , Peptides/metabolism , Phosphorylation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Engineering , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thermotoga maritima/genetics , Thermotoga maritima/metabolismABSTRACT
The increased worldwide awareness of seasonal and pandemic influenza, including pandemic H1N1 virus, has stimulated interest in the development of economic platforms for rapid, large-scale production of safe and effective subunit vaccines. In recent years, plants have demonstrated their utility as such a platform and have been used to produce vaccine antigens against various infectious diseases. Previously, we have produced in our transient plant expression system a recombinant monomeric hemagglutinin (HA) protein (HAC1) derived from A/California/04/09 (H1N1) strain of influenza virus and demonstrated its immunogenicity and safety in animal models and human volunteers. In the current study, to mimic the authentic HA structure presented on the virus surface and to improve stability and immunogenicity of the HA antigen, we generated trimeric HA by introducing a trimerization motif from a heterologous protein into the HA sequence. Here, we describe the engineering, production in Nicotiana benthamiana plants, and characterization of the highly purified recombinant trimeric HA protein (tHA-BC) from A/California/04/09 (H1N1) strain of influenza virus. The results demonstrate the induction of serum hemagglutination inhibition antibodies by tHA-BC and its protective efficacy in mice against a lethal viral challenge. In addition, the immunogenic and protective doses of tHA-BC were much lower compared with monomeric HAC1. Further investigation into the optimum vaccine dose and/or regimen as well as the stability of trimerized HA is necessary to determine whether trimeric HA is a more potent vaccine antigen than monomeric HA.
Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/administration & dosage , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , Animals , Antibodies, Bacterial/blood , Disease Models, Animal , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/isolation & purification , Influenza A Virus, H1N1 Subtype/genetics , Influenza Vaccines/administration & dosage , Mice, Inbred BALB C , Orthomyxoviridae Infections/immunology , Plants, Genetically Modified/genetics , Protein Engineering , Protein Multimerization , Survival Analysis , Nicotiana/genetics , Treatment Outcome , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
The chemical modification of a cysteinyl residue of D57C CheY by the addition of a phosphonomethyl group, (HO)(2)P(O)-CH(2)-, is described. This modification produces a nonlabile analog of an aspartyl phosphate residue in the active form of CheY. The chemically modified protein, phosphono-CheY, is suitable for structural and functional studies. An extensive discussion of the synthetic methodology and purification strategy is presented. A detailed protocol is given.
