RESUMO
It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.
Assuntos
Processamento de Proteína , Animais , Humanos , HidróliseRESUMO
Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to express cytotoxic proteins, to segmentally modify or label proteins, to cyclize proteins or peptides, to study structure-activity relationships and to generate reactive polypeptide termini in expressed proteins for an expanding list of chemoselective reactions, including protein ligation.
Assuntos
Biotecnologia/métodos , Processamento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/químicaRESUMO
Two genes, designated hpyA1 and hpyA2, have been cloned and sequenced from Pyrococcus strain GB-3a. They are predicted to encode proteins (HPyA1 and HPyA2, respectively) that are approx. 60% identical to the histones HMf and HMt, characterized from methanogenic Archaea. These archaeal histones also contain the amino-acid sequences, conserved in eukaryotic H4 histones, that are thought to interact directly with DNA.
Assuntos
Archaea/genética , Proteínas Arqueais , Proteínas de Bactérias/genética , Histonas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Proteínas de Ligação a DNA/genética , Dados de Sequência MolecularRESUMO
Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Brugia Malayi/enzimologia , Quitinases/genética , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de Ligação , Animais , Brugia Malayi/genética , Proteínas de Transporte/genética , Quitinases/metabolismo , Clonagem Molecular , Ativação Enzimática , Escherichia coli , Proteínas Ligantes de Maltose , Microfilárias/enzimologia , Microfilárias/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.
Assuntos
Vetores Genéticos , Processamento de Proteína Pós-Traducional , Processamento de Proteína , Proteínas Recombinantes/isolamento & purificação , ATPases Vacuolares Próton-Translocadoras , Marcadores de Afinidade , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Quitina , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose , Métodos , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/metabolismo , TemperaturaRESUMO
Two repeated sequences, plasmids pOV8 and pOV26, were cloned and characterized from the filarial parasite Onchocerca volvulus. Both clones can be used to distinguish O. volvulus DNA from other Onchocerca species or other nematodes by restriction fragment length polymorphisms, but neither clone can differentiate between DNA from savanna (Mali) or forest (Ivory Coast) O. volvulus isolates. DNA from one O. volvulus infective larva can be detected by both clones in dot blot hybridization assays. Neither clone cross hybridizes with DNA from host or vector species (human or simuliid, respectively). pOV26 is a member of an interspersed repeated DNA family composed of at least 100 members, and is only observed in the genus Onchocerca. Repeated DNA clone pOV8 cross reacts with DNA from other parasitic filarial nematodes, and is also present in at least 100 copies per O. volvulus genome. pOV26 is a potential tool in the diagnosis of human onchocerciasis, since it is specific for the genus Onchocerca. In the future, we plan to look for regions of these repeated sequences which may serve as a basis for the development of probes to discriminate among Onchocerca species and strains using a simple dot hybridization test.
Assuntos
DNA/análise , Onchocerca/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Clonagem Molecular , Côte d'Ivoire , Enzimas de Restrição do DNA , Eletroforese em Gel de Ágar , Genes , Larva , Mali , Hibridização de Ácido Nucleico , Onchocerca/classificação , Oncocercose/diagnóstico , Plasmídeos , Simuliidae , Especificidade da EspécieRESUMO
We report here the development of in situ hybridization and immunohistochemistry protocols which permit the histological identification of gene expression of a cloned antigen of Onchocerca volvulus, OI5, in the parasite. Skin nodules containing female adult worms were fixed in a modified Carnoy's fixative and embedded in paraffin. Histological staining of tissue sections revealed uniformly excellent morphology and RNA preservation. To localize mRNA by in situ hybridization, tissue sections were incubated with biotin-labeled pOI5, the plasmid containing the genomic sequence of the antigen, and hybridization signals were histochemically visualized using a streptavidin-enzyme conjugate and chromogenic substrates. The protein antigen was localized immunohistochemically by incubating the sections with specific antibodies prepared against a recombinant fusion protein containing the OI5 sequence (OI3), and visualized via a secondary antibody-biotin-enzyme conjugate procedure. The results reported here showed distinct localization of the OI5 mRNA and OI3 antigen in specific cellular and tissue regions of the adult parasite, and in microfilariae located within the uteri and in the surrounding host tissue. The specificity and high sensitivity of these histological detection methods should be generally applicable for the characterization of gene expression in the filarial parasite, particularly the insect-borne, infective filarial larvae, which are severely limited in quantity.
Assuntos
Expressão Gênica , Técnicas Histológicas , Onchocerca/genética , Animais , Antígenos de Helmintos/genética , DNA/metabolismo , Sondas de DNA , Feminino , Humanos , Hibridização de Ácido Nucleico , Pele/parasitologiaRESUMO
A 110 kDa Plasmodium knowlesi antigen, termed PK110, has been identified on the basis of messenger RNA abundance in late schizonts. Most Plasmodium genes previously cloned have been identified by immune sera, which have selected immunodominant antigens composed of repeating epitopes. Although PK110 was not selected by immune sera, it also contains amino acid repeats, indicating that this structure may be a common feature of malarial proteins. Determination of 296 codons in the PK110 gene revealed the presence of thirteen tandem repeats of twelve amino acids whose consensus sequence is E E T Q K T V E P E Q T. A termination codon interrupts the fourteenth repeat, indicating that these repeats are at the C-terminus of the protein. Indirect immunofluorescence experiments with sera raised against the lambda gt11 fusion protein indicate that PK110 is present in intra-erythrocytic late schizonts. Cloned PK110 is recognized by Gambian sera, and shares epitopes with Plasmodium ovale. PK110 does not cross react immunologically or by DNA hybridization with Plasmodium falciparum.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Clonagem Molecular , Plasmodium/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Sequência de Bases , Reações Cruzadas , DNA/análise , Epitopos/análise , Epitopos/genética , Gâmbia , Genes , Macaca mulatta , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmodium/genética , Plasmodium falciparum/imunologia , RNA Mensageiro/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
Cellular immune responses play a major role in lymphatic filarial infections. To further our understanding of the host-parasite interaction, we investigated T-cell stimulation by purified filarial recombinant antigens in peripheral blood mononuclear cells derived from filarial-infected individuals. One of a subset of cloned Brugia malayi antigens involved in the humoral immune response to filarial infection was found to be a T-cell-stimulating antigen. The fusion protein encoded by clone lambda Bm19 induced proliferation of human T cells in a parasite-specific, antigen dose-dependent manner. The deduced amino acid sequence from this cloned region revealed 4 predicted T-cell recognition sites. The lambda Bm19 DNA sequence hybridizes to a 3-kb transcript, and in situ mRNA hybridization analyses of the adult female worm demonstrated that this gene is expressed in developing uterine microfilariae. The native parasite protein is present in several developmental stages since clone lambda Bm19 was initially identified with antiserum directed against the infective larval stage; this protein is therefore a potential target for the host's immune system.
Assuntos
Antígenos de Helmintos/genética , Brugia/genética , Ativação Linfocitária , RNA Mensageiro/genética , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/imunologia , Sequência de Bases , Brugia/imunologia , Clonagem Molecular , DNA/análise , Feminino , Genes de Imunoglobulinas , Humanos , Masculino , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/biossínteseRESUMO
The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system. Furthermore, the M. xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.
Assuntos
Proteínas de Bactérias/isolamento & purificação , DNA Topoisomerases Tipo II/química , Mycobacterium xenopi/enzimologia , Processamento de Proteína , Proteínas de Bactérias/química , Proteínas de Transporte/genética , Clonagem Molecular , DNA Girase , DNA Topoisomerases Tipo II/genética , Escherichia coli , Lectinas de Ligação a Manose , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/enzimologia , Temperatura , Tiorredoxinas/química , Tropomiosina/químicaRESUMO
We examined the reactivity of human sera with recombinant microfilarial chitinase and with the antigenic determinant on the native parasite molecule identified by monoclonal antibody (MAb) MF1. In Brugian filariasis, the MF1 epitope is preferentially recognized by residents of endemic areas who remain amicrofilaremic and asymptomatic despite lifelong exposure to filarial worms. Reactivity with filarial chitinase and its MF1 epitope inversely correlates with microfilaremia levels in Bancroftian filariasis and is associated with a prolonged amicrofilaremic state following a single course of treatment with diethylcarbamazine. Chitinase does not appear to be a target of human antibodies that promote the adherence of cells to microfilariae, even though MAb MF1 itself promotes antibody-dependent, cell-mediated cytotoxic (ADCC) reactions that kill microfilariae in vitro. Such ADCC reactions are most often mediated by sera from amicrofilaremic patients with chronic elephantiasis that contain low or undetectable levels of IgG antibodies to chitinase. In contrast, antibodies to the MF1 epitope on this microfilarial stage-specific antigen are mostly present in amicrofilaremic donors without clinical lymphatic disease. These observations indicate that antibodies to the MF1 epitope of microfilarial chitinase reflect some degree of immune resistance to microfilaremia in a subgroup of patients with asymptomatic lymphatic filariasis. The amicrofilaremic state of individuals with chronic lymphatic disease appears to be mediated by reactivity to a different parasite antigen(s).
Assuntos
Antígenos de Helmintos/imunologia , Brugia Malayi/imunologia , Quitinases/imunologia , Filariose/imunologia , Wuchereria bancrofti/imunologia , Adulto , Animais , Citotoxicidade Celular Dependente de Anticorpos , Brugia Malayi/enzimologia , Epitopos/imunologia , Humanos , Microfilárias/enzimologia , Wuchereria bancrofti/enzimologiaRESUMO
The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Modelos Animais de Doenças , Muridae , Onchocerca/crescimento & desenvolvimento , Oncocercose/imunologia , Animais , Criopreservação , Cultura em Câmaras de Difusão , Gerbillinae , Imunização , Larva/crescimento & desenvolvimento , Larva/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Onchocerca/imunologia , Oncocercose/parasitologia , Ratos , Ratos EndogâmicosRESUMO
The study of enzymes isolated from organisms inhabiting unconventional ecosystems has led to the realization that biocatalysis need not be constrained to mild conditions and can be considered at pH's, temperatures, pressures, ionic and solvent environments long thought to be destructive to biomolecules. Parallel to this, it has been demonstrated that even conventional enzymes will catalyze reactions in solvents other than water. However, the intrinsic basis for biological function under extreme conditions is only starting to be addressed, as are associated applications. This was the focus of a recent NSF/NIST-sponsored workshop on extremozymes. Given the information acquired from the study of extremozymes, modification of enzymes to improve their ranges of stability and activity remains a possibility. Ultimately, by expanding the range of conditions suitable for enzyme function, new opportunities to use biocatalysis will be created.
Assuntos
Ecossistema , Enzimas/metabolismo , Temperatura , Catálise , Concentração de Íons de Hidrogênio , Concentração Osmolar , Pressão , SolventesAssuntos
Archaea/enzimologia , DNA Polimerase Dirigida por DNA/química , Bacillus/enzimologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Dados de Sequência Molecular , Alinhamento de Sequência , Sulfolobus/enzimologia , Thermus/enzimologiaAssuntos
Proteínas de Bactérias/genética , Mathanococcus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Helicases/química , DNA Helicases/genética , Endonucleases/química , Temperatura Alta , Mathanococcus/enzimologia , Mycobacterium/enzimologia , Mycobacterium/genética , Engenharia de Proteínas , Processamento de Proteína/genética , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genéticaRESUMO
Inteins are intervening sequences that splice as proteins, not RNA. InBase, the New England Biolabs Intein Database (http://www.neb. com/neb/inteins.html), is a comprehensive on-line database that includes the Intein Registry, along with detailed information about each intein and its host protein, tabulated comparisons and a comprehensive bibliography including papers in press.
Assuntos
Bases de Dados Factuais , Proteínas de Drosophila , Processamento de Proteína , Proteínas/química , Proteínas/metabolismo , Animais , Sequência Consenso , Endonucleases/química , Endonucleases/metabolismo , Proteínas Hedgehog , Armazenamento e Recuperação da Informação , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Internet , New England , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Formation of an internal (thio)ester bond activates numerous in vivo protein autoprocessing pathways including pyruvoyol group synthesis, autoproteolysis, protein splicing, enzyme activation and protein targeting. Structural analysis of precursors, intermediates and products is fine tuning our understanding of the mechanisms of these reactions.
Assuntos
Ésteres , Proteínas/química , Compostos de Sulfidrila , Cisteína , Ativação Enzimática , Enzimas/química , Enzimas/metabolismo , Modelos Químicos , Serina , TreoninaRESUMO
InBase, the Intein Database (http://www.neb.com/neb/inteins.html ), is a comprehensive on-line resource that includes the Intein Registry. Inteins are protein splicing elements that mediate a self-catalytic protein splicing reaction. InBase presents general information as well as detailed data for each intein, including tabu-lated comparisons and a comprehensive bibliography.