FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Characterization of the Regions Involved in the Calcium-Induced Folding of the Intrinsically Disordered RTX Motifs from the Bordetella pertussis Adenylate Cyclase Toxin

Repeat in toxin (RTX) motifs are nonapeptide sequences found among numerous virulence factors of Gram-negative bacteria. In the presence of calcium, these RTX motifs are able to fold into an idiosyncratic structure called the parallel β-roll. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, is one of the best-characterized RTX cytolysins. CyaA contains a C-terminal receptor domain (RD) that mediates toxin binding to the eukaryotic cell receptor. The receptor-binding domain is composed of about forty RTX motifs organized in five successive blocks (I to V). The RTX blocks are separated by non-RTX flanking regions of variable lengths. It has been shown that block V with its N- and C-terminal flanking regions constitutes an autonomous subdomain required for the toxicity of CyaA. Here, we investigated the calcium-induced biophysical changes of this subdomain to identify the respective contributions of the flanking regions to the folding process of the RTX motifs. We showed that the RTX polypeptides, in the absence of calcium, exhibited the hallmarks of intrinsically disordered proteins and that the C-terminal flanking region was critical for the calcium-dependent folding of the RTX polypeptides, while the N-terminal flanking region was not involved. Furthermore, the secondary and tertiary structures were acquired concomitantly upon cooperative binding of several calcium ions. This suggests that the RTX polypeptide folding is a two-state reaction, from a calcium-free unfolded state to a folded and compact conformation, in which the calcium-bound RTX motifs adopt a β-roll structure. The relevance of these results to the toxin physiology, in particular to its secretion, is discussed.     

Proteolysis and synthetic strategy of human G-CSF in Escherichia coli BL21(DE3)

We report for the first time that the C-terminal region of hG-CSF suffers from proteolytic degradation when human granulocyte colony-stimulating factor (hG-CSF) is directly expressed in Escherichia coli BL21(DE3). It is believed that the rapid proteolysis occurs at the C-terminus of hG-CSF that is very easily exposed to E. coli protease(s) during a short period following protein synthesis and prior to completion of the formation of the inclusion body. The recombinant hG-CSF that is expressed with an N-terminal fusion partner is effectively protected from the proteolysis. It seems that since the N-terminus of hG-CSF is located very close to the C-terminus, the presence of the N-terminal fusion partner masks the C-terminal region of hG-CSF and protects it from proteolytic degradation by E. coli protease(s). Furthermore, the solubility of hG-CSF markedly increased in E. coli cytoplasm when a stress-responsive and aggregation-resistant protein, i.e. aspartate carbamoyl-transferase catalytic chain (PyrB) was used as a novel N-terminal fusion partner proteins.     

Mutational Analysis of the Amino Acid Residues Essential for the cis and trans Cleavage Activity of the Potato Virus Y 50-kDa Protease

To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.     

Expression of a functional 78,000 dalton mammalian flavoprotein,NADPH-cytochrome P-450 oxidoreductase,in Escherichia coli

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2′5′-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5–10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 μmol of cytochrome c reduced/mg reductase/min at 22 °C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.     

Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21

The manganese (Mn)-oxidizing protein (MopA) from Erythrobacter sp. strain SD21 is part of a unique enzymatic family that is capable of oxidizing soluble Mn(II). This enzyme contains two domains, an animal heme peroxidase domain, which contains the catalytic site, followed by a C-terminal calcium binding domain. Different from the bacterial Mn-oxidizing multicopper oxidase enzymes, little is known about MopA. To gain a better understanding of MopA and its role in Mn(II) oxidation, the 238-kDa full-length protein and a 105-kDa truncated protein containing only the animal heme peroxidase domain were cloned and heterologously expressed in Escherichia coli. Despite having sequence similarity to a peroxidase, hydrogen peroxide did not stimulate activity, nor was activity significantly decreased in the presence of catalase. Both pyrroloquinoline quinone (PQQ) and hemin increased Mn-oxidizing activity, and calcium was required. The K_m for Mn(II) of the full-length protein in cell extract was similar to that of the natively expressed protein, but the K_m value for the truncated protein in cell extract was approximately 6-fold higher than that of the full-length protein, suggesting that the calcium binding domain may aid in binding Mn(II). Characterization of the heterologously expressed MopA has provided additional insight into the mechanism of bacterial Mn(II) oxidation, which will aid in understanding the role of MopA and Mn oxidation in bioremediation and biogeochemical cycling.     

Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ~97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ~45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.     

A possible cause of heterogeneity of mammalian apurinic/apyrimidinic endonuclease

• 1.1. Mammalian major apurinic/apyrimidinic (AP) endonuclease, APEX nuclease (M_r 35.4 kDa) was purified from HeLa cells. A hybrid protein (M_r 36.4 kDa), which was expressed in BW2001 strain cells of E. coli, comprising human APEX nuclease headed by 10 additional amino acids was also purified.
• 2.2. The purified preparations were frequently associated with 31-, 33- and 35-kDa peptides having AP endonuclease activity.
• 3.3. The 33- and 35-kDa peptides were suggested to be formed from the hybrid protein or APEX nuclease during their purification processes by proteolytic cleavage with subtilisin-like protease. The 31-kDa peptide was thought to be produced by chemical cleavage of the aspartyl-prolyl bond of APEX nuclease.
• 4.4. The results support the notion that some of AP endonuclease heterogeneity based on the molecular weight difference are caused by proteolytic (and chemical) cleavage of a species of AP endonucleases during the extraction and purification.

     

C-terminal processing of tyrosinase is responsible for activation of Pholiota microspora proenzyme

Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal.     

Structural modifications of outer membrane vesicles to refine them as vaccine delivery vehicles

In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles.     

Bordetella Adenylate Cyclase Toxin Promotes Calcium Entry into Both CD11b+ and CD11b? Cells through cAMP-dependent L-type-like Calcium Channels

Adenylate cyclase toxin (ACT), a 200 kDa protein, is an essential virulence factor for Bordetella pertussis, the bacterium that causes whooping cough. ACT is a member of the pore-forming RTX (repeats-in-toxin) family of proteins that share a characteristic calcium-binding motif of Gly- and Asp-rich nonapeptide repeats and a marked cytolytic or cytotoxic activity. In addition, ACT exhibits a distinctive feature: it has an N-terminal calmodulin-dependent adenylate cyclase domain. Translocation of this domain into the host cytoplasm results in uncontrolled production of cAMP, and it has classically been assumed that this surge in cAMP is the basis for the toxin-mediated killing. Several members of the RTX family of toxins, including ACT, have been shown to induce intracellular calcium increases, through different mechanisms. We show here that ACT stimulates a raft-mediated calcium influx, through its cAMP production activity, that activates PKA, which in turn activates calcium channels with L-type properties. This process is shown to occur both in CD11b⁺ and CD11b⁻ cells, suggesting a common mechanism, independent of the toxin receptor. We also show that this ACT-induced calcium influx does not correlate with the toxin-induced cytotoxicity.     

Proteins that bind calcium in a phospholipid-dependent manner

Three proteins (Mr = 64K, 32K, and 22K) that bind to phospholipids in a calcium-dependent manner were purified from bovine brain. The calcium-binding properties of these proteins were investigated by equilibrium dialysis and by gel filtration chromatography. The 64- and 32-kDa proteins were found to have calcium- and phospholipid-binding properties strikingly similar to those of protein kinase C [Bazzi, M.D., & Nelsestuen, G.L. (1990) Biochemistry 29, 7624]. The free proteins bound limited divalent metal ion even at 200 microM calcium. However, they bound eight to nine calcium ions per protein in the presence of membranes containing acidic phospholipids. The calcium concentrations needed for protein-phospholipid binding were different for these two proteins and were strongly influenced by the phospholipid composition of the vesicles; vesicles of higher phosphatidylserine content required lower concentrations of calcium for protein-membrane association. These properties described a general type of calcium-interacting system where simultaneous interaction of all three components (protein, phospholipids, and calcium) is required. The free proteins may provide only partial coordinate bonds to each calcium ion, but complete calcium-binding sites could be generated at the protein-phospholipid interface. In contrast to the 64- and 32-kDa proteins, the 22-kDa protein bound similar amounts of calcium (two to three ions/protein) in the presence or the absence of phospholipids. The 22-kDa protein had the lowest affinity for phospholipid and the highest affinity for calcium of the three proteins tested. Thus, calcium-dependent phospholipid-binding proteins consist of several types. For example, the 64- and 32-kDa proteins appear to be quite abundant and may even function as a calcium buffer to modulate signaling events.     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k_cat/K_m) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15°C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100°C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the ¹H, ¹³C and ¹⁵N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. ¹H–¹⁵N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.     

Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens

The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX) family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.