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The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.
Perchloric acid-soluble protein (PSP) may play an important role in the regulation of cellular physiological functions because it has been highly conserved throughout evolution; however, this role has not been well elucidated. In previous reports, we suggested that PSP regulates cell proliferation. In this study, we examined the effect of PSP expression on proliferation of the normal rat kidney cell line NRK-52E, the rat hepatocyte cell line RLN-10, and the rat hepatoma cell line dRLh-84. Cells transfected with pcDNA-sense-PSP (pcDNA-S-PSP) over-expressed PSP mRNA and protein, and cell proliferation of the transfected cells was suppressed compared with that of cells transfected with pcDNA-empty (pcDNA-E). Cell viability of pcDNA-S-PSP-transfected cells was similar to that of pcDNA-E-transfected cells. Thus, over-expression of PSP suppresses cell proliferation without any influence on cell viability. These findings are the first to report an inhibitory activity of PSP on cell proliferation.
Dentin matrix protein 1 (DMP1) is an extracellular matrix noncollagenous protein (NCP) initially isolated from dentin and now found to be present in calcified tissues like calvaria and long bone. The characteristic feature of DMP1 is that it contains a large number of acidic domains and has properties which implicate it as a key participant in regulating matrix mineralization. The level of DMP1 in the tissue is sparse and it is not easily isolated from dentin because it copurifies with other dentin NCPs. The exact function of DMP1 is not known and this is due to the inherent difficulty of obtaining enough protein from the mineralized tissues. In order to understand the physiologic role for DMP1 during the formation of mineralized tissues we have produced milligram quantities of recombinant DMP1 in E. coli. The objective of this work was: (1) to prepare unmodified apoprotein so that it could be used for studying the function of DMP1; and (2) to prepare polyclonal antibody against the recombinant DMP1 antigen. The DMP1 polyclonal antibody did not cross-react with other NCPs present in dentin or with bone acidic glycoprotein-75 (BAG-75) present in the bone matrix, confirming the specificity of this antibody and thus making it a valuable tool to determine the in vivo function of DMP1.
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Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species. BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult. To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita). Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences. No protein was detected after induction of full-length C1 expression in Escherichia coli. However, deletion of an amino-terminal arginine-rich sequence facilitated expression. C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens. The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds. Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting. The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.
A safer and more effective vaccine than the previously developed live attenuated vaccine is needed for combating Francisella tularensis, a highly infectious bacterial pathogen. To search for potential candidates for inclusion in a new vaccine, we characterized the proteins present in the culture filtrates of a virulent recent clinical isolate and the attenuated live vaccine strain of F. tularensis using a proteomic approach. We identified a total of 12 proteins; among these, catalase-peroxidase was much more abundant in the culture filtrate of the virulent clinical isolate, whereas bacterioferritin was more abundant in the culture filtrate of the live vaccine strain. Streptolysin O treatment of infected human macrophages indicated that catalase-peroxidase and the heat shock protein GroEL are released intracellularly by actively growing F. tularensis. Mice immunized with F. tularensis developed significant cell-mediated immune responses to catalase-peroxidase, the heat shock protein GroEL, and bacterioferritin as measured by splenic lymphocyte proliferation and gamma interferon production. Finally, we expressed the major culture filtrate proteins that are promising vaccine candidates in Escherichia coli at high levels in soluble form to facilitate study of their immunobiology and potential role in vaccines.
BACKGROUND: In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. RESULTS: The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. CONCLUSION: A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family-specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.
FMLP-receptor DNA was expressed in Escherichia coli. The expressed product could specifically bind FMLP. This is the first-reported expression of a functional FMLP receptor in Escherichia coli. We confirm that receptor glycosylation is not essential for ligand binding. A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.
The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.
The p55 gag gene of simian immunodeficiency virus macaque strain (SIVmac) and the core p27 gag component linked to a synthetic AUG codon have been cloned into adenovirus type 5 vectors to generate either viable E3-replacement or defective E1-replacement viruses. The viruses express the expected SIV proteins in both human and, for the non-defective viruses, monkey cells. A considerable proportion of the p55 produced is exported from the infected cell. These viruses should prove useful both in studies of the immune response to SIV and as components of candidate vaccines aimed specifically at provoking cytotoxic T cell responses.
An estrogen receptor beta (ERbeta) cDNA fragment was amplified by RT-PCR of total RNA extracted from liver and ovary of immature channel catfish. This cDNA fragment was used to screen an ovarian cDNA library made from an immature female fish. A clone was obtained that contained an open reading frame encoding a 575-amino-acid protein with a deduced molecular weight of 63.9 kDa. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of channel catfish ERbeta on the basis of 25 full-length teleost and tetrapod ER sequences. The consensus tree obtained indicated the existence of two major vertebrate ER subtypes, alpha and beta. Within each subtype, and in accordance with established phylogenetic relationships, teleost and tetrapod ER were monophyletic confirming the results of a previous analysis (Z. Xia et al., 1999, Gen. Comp. Endocrinol. 113, 360-368). Extracts of COS-7 cells transfected with channel catfish ERbeta cDNA bound estrogen with high affinity (K(d) = 0.21 nM) and specificity. The affinity of channel catfish ERbeta for estrogen was higher than previously reported for channel catfish ERalpha. As determined by qualitative RT-PCR, the tissue distributions of ERalpha and ERbeta were similar but not identical. Both ER subtypes were present in ovary and testis. ERalpha was found in all other tissues examined from juvenile and mature fish of both sexes. ERbeta was also found in most tissues except, in most cases, whole blood and head kidney. Interestingly, the pattern of expression of ER subtypes in head kidney always corresponded to the pattern in whole blood. In conclusion, we isolated a channel catfish ERbeta with ligand-binding affinity and tissue expression patterns different from ERalpha. Also, we confirmed the validity of our previously proposed general classification scheme for vertebrate ER into alpha and beta subtypes and within each subtype, into teleost and tetrapod clades.
Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.
We recently found that the larger parts of the endocytic proteins epsin 1 and AP180 consist of an unstructured polypeptide chain. As a result these segments are completely heat-stable without loss of their functional properties. We have taken advantage of this fact and developed a combined heat lysis and pre-purification procedure after expressing the disordered domains in E. coli. This results in the irreversible denaturation and precipitation of the majority of bacterial proteins. The bacteria are resuspended in a non-denaturing buffer, heated in a boiling water bath and shock-cooled. We demonstrate that this procedure compared to conventional lysis improves both yield and quality of the purified protein.
Antimicrobial peptides or proteins (AMPs) are proved to be one of the most important humoral factors to resist pathogen infection. As an antimicrobial protein, crustin had been described in invertebrates as a component of the innate immune system. A crustin-like gene (CruFc) was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5'-RACE PCR. The full-length cDNA consists of 523 with 405 bp open reading frame encoding 134 amino acids and the deduced peptide contains a putative signal peptide of 17 amino acids. The sequence also contains a whey-acidic protein (WAP) domain at the C-terminal. Transcripts of CruFc were mainly detected in haemocytes and gill by RT-PCR analysis. In addition, another full-length cDNA named CshFc was also cloned from haemocytes of Chinese shrimp and its inferred amino acid sequence lacks the WAP-type 'four-disulfide core' domain. The fusion proteins containing CruFc and CshFc were, respectively, produced and the antimicrobial assays revealed that the recombinant CruFc could inhibit the growth of gram-positive bacteria in vitro but the recombinant CshFc could not inhibit at the same conditions. The difference of antimicrobial activity between recombinant CruFc and CshFc provides the evidence that the four-disulfide core domain of crustin may play an important role in its biological function.
A novel metallocarboxypeptidase inhibitor was isolated from the medical leech Hirudo medicinalis. Amino acid sequence analysis provided a nearly complete primary structure. which was subsequently verified and completed by cDNA cloning using reverse transcriptase-polymerase chain reaction/rapid amplification of cDNA end techniques. The inhibitor, called LCI (leech carboxypeptidase inhibitor), is a cysteine-rich polypeptide composed of 66 amino acid residues. It does not show sequence similarity to any other protein except at its C-terminal end. In this region, the inhibitor shares the amino acid sequence -Thr-Cys-X-Pro-Tyr-Val-X with Solanacea carboxypeptidase inhibitors, suggesting a similar mechanism of inhibition where the C-terminal tail of the inhibitor interacts with the active center of metallocarboxypeptidases in a substrate-like manner. This hypothesis is supported by the hydrolytic release of the C-terminal glutamic acid residue of LCI after binding to the enzyme. Heterologous overexpression of LCI in Escherichia coli, either into the medium or as an intracellular thioredoxin fusion protein, yields a protein with full inhibitory activity. Both in the natural and recombinant forms, LCI is a tightly binding, competitive inhibitor of different types of pancreatic-like carboxypeptidases, with equilibrium dissociation constants Ki of 0.2-0.4 x 10(-9) M for the complexes with the pancreatic enzymes A1, A2, and B and plasma carboxypeptidase B. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicate that recombinant LCI is a compactly folded globular protein, stable to a wide range of pH and denaturing conditions.
Polyclonal antibodies were generated in rabbits by delivery to skin of gold particles coated with mammalian expression vectors encoding a cytoplasmic (beta-galactosidase) or a nuclear (L1 capsid of cottontail rabbit papillomavirus) protein. One primary and one booster immunization of 30 micrograms DNA per rabbit yielded specific antisera with titers from 1:24 000 to 1:120 000 in each of eight rabbits, as detected by ELISA and Western blot analysis. Genetic immunization requires relatively small amounts of DNA, eliminates the need to purify the protein immunogen, and does not require irritating adjuvants.
Toxic shock syndrome (TSS) is caused by the staphylococcal superantigen, TSST-1. The MHC class II binding domain of TSST-1 containing a conserved sequence with other related staphylococcal enterotoxins, comprising TSST-1 residues 47-64 [(T(47-64)], was expressed as a fusion protein with either glutathione-S-transferase (GST(47-64)), filamentous phage coat protein (pIII(47-64)), or E. coli outer membrane porin protein (OprF(47-64)), or synthesized as a peptide conjugated to bovine serum albumin, BSA(47-64). GST(47-64), OprF(47-64) and BSA(47-64), but not pIII(47-64), all induced high-titer T(47-64)-specific antibodies in Balb/c mice. However, only anti-GST(47-64) antibodies inhibited (125)I-TSST-1 binding to MHC class II and abrogated TSST-1-induced T cell mitogenesis and TNFalpha secretion in human peripheral blood mononuclear cells. Purified GST(47-64) also inhibited (125)I-TSST-1 binding in a dose-dependent manner. These findings suggest that GST(47-64) may have potential as a recombinant peptide vaccine or TSST-1 receptor inhibitor against TSS.
New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics. beta-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses beta-lactam resistance. beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified. The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major beta-lactamase of M. tuberculosis is a class A beta-lactamase with predominant penicillinase activity. A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present.