RESUMO
The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by "reversion" of Tyr+1 to Cys.
Assuntos
Magnetospirillum/metabolismo , Splicing de RNA , Alanina/química , Processamento Alternativo , Catálise , Cisteína/química , Modelos Genéticos , Mutação , Fenilalanina/química , Polimorfismo Genético , Processamento de Proteína , Estrutura Terciária de Proteína , Serina/química , Tirosina/químicaAssuntos
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
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
Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.
Assuntos
Processamento Alternativo , Proteínas de Bactérias , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cisteína/química , Processamento de Proteína Pós-Traducional , Serina/química , Alanina/química , Catálise , Linhagem Celular , Clonagem Molecular , Mathanococcus/genética , Mathanococcus/metabolismo , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Polimorfismo Genético , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.
Assuntos
Histidina/química , Mathanococcus/enzimologia , Processamento de Proteína , Asparagina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Estrutura Molecular , Mutação , Fosfotransferases (Aceptores Pareados)/genética , Fosfotransferases (Aceptores Pareados)/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de ProteínaRESUMO
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.
Assuntos
Bases de Dados Factuais , Processamento de Proteína/genética , InternetRESUMO
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
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
Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.
Assuntos
Fragmentos de Peptídeos/metabolismo , Processamento de Proteína , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Enzimas de Restrição do DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Choque Térmico/genética , Proteínas Ligantes de Maltose , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Precursores de Proteínas/biossíntese , Precursores de Proteínas/síntese química , Precursores de Proteínas/genética , Processamento de Proteína/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/metabolismo , Tropomiosina/genéticaAssuntos
Proteínas de Drosophila , Proteínas de Insetos/fisiologia , Processamento de Proteína Pós-Traducional , Processamento de Proteína , Sequência de Aminoácidos , Evolução Molecular , Proteínas Hedgehog , Proteínas de Insetos/genética , Dados de Sequência Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Using two recombinant filarial protein Ags and keyhole limpet hemocyanin, we sensitized T cells from uninfected, nonatopic individuals in such a manner that they were able to provide help for the selective induction of an Ag-specific Ab response. IL-2 and IL-4 were shown to be critical for sensitizing the T cells; once sensitized, these T cells could provide the necessary signals for B cells to produce Ag-specific Abs, provided that IL-4 (or IL-2) was supplied exogenously. Primary exposure of T cells to IFN-gamma, but not to IL-12, prevented the Ag-sensitized T cells from helping B cells to produce specific Abs, apart from the IgG2 isotype. These data suggest that Ab-producing B cells of a defined Ag specificity and isotype can be generated differentially after in vitro priming of human T cells by Ag, providing regulatory cytokines are also present.
Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Antígenos de Helmintos/imunologia , Linfócitos B/imunologia , Citocinas/fisiologia , Epitopos de Linfócito B/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/parasitologia , Diferenciação Celular/imunologia , Epitopos de Linfócito T/imunologia , Hemocianinas/imunologia , Humanos , Imunização , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Interferon gama/farmacologia , Interleucina-12/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Cooperação Linfocítica , Moluscos , Linfócitos T/citologia , Linfócitos T/parasitologiaRESUMO
The 198-amino-acid in-frame insertion in the gyrA gene of Mycobacterium xenopi is the smallest known naturally occurring active protein splicing element (intein). Comparison with other mycobacterial gyrA inteins suggests that the M. xenopi intein underwent a complex series of events including (i) removal of 222 amino acids that encompass most of the central intein domain, and (ii) addition of a linker of unrelated residues. This naturally occurring genetic rearrangement is a representative characteristic of the taxon. The deletion process removes the conserved motifs involved in homing endonuclease activity. The linker insertion represents a structural requirement, as its mutation resulted in failure to splice. The M. xenopi GyrA intein thus provides a paradigm for a minimal protein splicing element.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium xenopi/genética , Processamento de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ditiotreitol/farmacologia , Dados de Sequência Molecular , Mycobacterium xenopi/química , Mycobacterium xenopi/metabolismo , Análise de Sequência , TemperaturaRESUMO
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
We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996. Analysis of the 36 available intein sequences refines the previously described intein motifs and reveals the presence of another intein motif, Block H. Furthermore, analysis of the new inteins reshapes our view of the conserved splice junction residues, since three inteins lack the intein penultimate His seen in prior examples. Comparison of intein sequences suggests that, in general, (i) inteins present in the same location within extein homologs from different organisms are very closely related to each other in paired sequence comparison or phylogenetic analysis and we suggest that they should be considered intein alleles; (ii) multiple inteins present in the same gene are no more similar to each other than to inteins present in different genes; (iii) phylogenetic analysis indicates that inteins are so divergent that trees with statistically significant branches cannot be generated except for intein alleles.
Assuntos
Sequência de Aminoácidos , Sequência Conservada , Processamento de Proteína , Proteínas/química , Sequência Consenso , Enzimas/química , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , SoftwareRESUMO
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 results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.
Assuntos
Modelos Genéticos , Mutação , Precursores de Proteínas/genética , Processamento de Proteína/fisiologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Cisteína/farmacologia , DNA Polimerase Dirigida por DNA/genética , Hidroxilamina , Hidroxilaminas/farmacologia , Proteínas Ligantes de Maltose , Mutagênese Insercional , Processamento de Proteína/genética , Proteínas Recombinantes/genética , Tropomiosina/genéticaRESUMO
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.