RESUMO
Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16â¯mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.
Assuntos
Catepsina D/isolamento & purificação , Schistosoma mansoni/enzimologia , Animais , Ácido Aspártico Proteases/biossíntese , Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/isolamento & purificação , Ácido Aspártico Proteases/metabolismo , Catepsina D/biossíntese , Catepsina D/química , Catepsina D/metabolismo , Catepsinas/biossíntese , Catepsinas/química , Catepsinas/isolamento & purificação , Catepsinas/metabolismo , Cromatografia em Gel , Dimerização , Células HEK293 , Humanos , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ultracentrifugação/métodosRESUMO
Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.
Assuntos
Antivirais/farmacologia , Biologia Computacional , Cristalografia , Desenho de Fármacos , Genômica , Proteômica , Vírus de RNA/efeitos dos fármacos , RNA Polimerase Dependente de RNA , Replicação Viral/efeitos dos fármacos , Antivirais/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cooperação Internacional , Modelos Moleculares , Vírus de RNA/enzimologia , Vírus de RNA/patogenicidade , Vírus de RNA/fisiologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismoRESUMO
The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.
Assuntos
Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos de Imunoglobulinas/uso terapêutico , Neoplasias Experimentais/radioterapia , Radioimunoterapia , Animais , Células CHO , Bovinos , Cricetinae , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Transplante Heterólogo , Radioisótopos de Ítrio/uso terapêuticoRESUMO
A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Catálise , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , DNA Complementar , Expressão Gênica , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137-141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511-2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen-binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution.
Assuntos
Sequência de Bases , Colágeno/metabolismo , Fibronectinas/genética , Genes , Clonagem Molecular , Humanos , Hibridização de Ácido NucleicoRESUMO
Here we report on a monoclonal antibody (IST-9) which distinguishes between human cellular and plasma fibronectin. Using beta-galactosidase-fibronectin fusion proteins expressed in E. coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST-1 and IST-2), specific for the heparin-binding domain 5 of fibronectin.
Assuntos
Anticorpos Monoclonais , Epitopos/análise , Fibronectinas/análise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Escherichia coli/metabolismo , Fibronectinas/imunologia , Humanos , Splicing de RNA , beta-Galactosidase/metabolismoRESUMO
A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra a AIDS/efeitos adversos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Esquemas de Imunização , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Pan troglodytes , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas do Envelope Viral/genéticaRESUMO
A number of recent technological developments have greatly facilitated the genetic engineering of immunoglobulins. The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunised cells. The conversion of the rodent antibody into a humanized version is now well established. To develop these antibodies for clinical use has required the development of high level expression systems. For the expression of large multimeric glycoproteins, mammalian cell systems generally provide the highest levels of secreted product and therefore are the methods of choice for producing whole recombinant antibodies. Novel antigen-binding units have been developed by joining the two variable domains of an antibody into single-chain polypeptides. Such fragments can be produced in high yield by secretion from E. coli raising the prospect of bulk preparation of these antibody fragments for the development of low-cost immunopurification and assay reagents. Finally, the ability to screen for antigen binding by displaying immunoglobulin variable regions on the surface of filamentous bacteriaphages has opened up the possibility of bypassing the immune system to generate novel antibody specificities in vitro.
Assuntos
Anticorpos Monoclonais/genética , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Bactérias/genética , Células CHO , Cricetinae , Expressão Gênica , Genes de Imunoglobulinas/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genéticaRESUMO
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is essential for virus entry and the formation of multinucleated giant cells by cell fusion, one of the major virus-induced cytopathic effects. To study the effects of potential fusion inhibitors, a vaccinia virus recombinant expressing the envelope glycoprotein was generated and used to infect HeLa CD4+ cells. Syncytium induction was observed as early as 4 h postinfection and continued until the entire monolayer was fused. The N-terminus of the gp41 subunit of the HIV envelope protein is very hydrophobic, and appears to be involved in virus-induced membrane fusion. We synthesized several oligopeptide analogs of the N-terminal region of gp41 and determined their ability to inhibit HIV-induced cell fusion in CD4+ HeLa cells. A hexapeptide which was identical in amino acid sequence to the N-terminus of gp41 was found to completely inhibit cell fusion, whereas peptides with altered sequences showed reduced inhibitory activity. These peptides had no effect on protein synthesis, processing, or transport to the cell surface, and showed no signs of toxicity to cells even at very high concentrations. These results indicate that oligopeptides which are homologous to the fusion peptide of HIV inhibit virus-induced cytopathology, and should be evaluated further as potential antiviral agents.
Assuntos
Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/fisiologia , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Transporte Biológico , Imunofluorescência , Produtos do Gene env/biossíntese , Produtos do Gene env/metabolismo , Células Gigantes , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Fusão de Membrana , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Vaccinia virus/genéticaRESUMO
A recent isolate of human immunodeficiency virus type 2 (HIV-2) designated HIV-2ST is deficient in its ability to cause the typical cytopathic effects of HIV infection. The pathogenic potential of HIV-2 in inducing human disease may be less than that of HIV-1, and it is of particular interest to establish the basis for the reduced cytopathogenicity of this isolate in vitro. Utilizing recombinant vaccinia viruses (rVV) carrying the envelope genes (env) of HIV-2ST or those of fully cytopathic HIV-1 or HIV-2 isolates, we have investigated envelope glycoprotein expression, processing, transport, and biological function. Radioimmunoprecipitation and polyacrylamide gel electrophoresis (RIP-PAGE) of rVV-infected cell lysates indicated that the proteins expressed by each recombinant were synthesized, processed, and recognized by specific antisera. Immunofluorescence studies showed that the recombinant env gene products of HIV-2ST and HIV-2ROD reach the cell surface and are retained there in similar amounts. Whereas cells expressing the HIV-1 or HIV-2ROD env gene products were found to undergo fusion with uninfected CD4+ cells, no syncytium formation was observed with three CD4+ cell lines exposed to the cells expressing the envelope glycoproteins of HIV-2ST on their surfaces; one CD4+ lymphoid cell line (SupT1) exhibited few very small syncytia in the presence of recombinant HIV-2ST envelope glycoproteins. The failure of the HIV-2ST envelope glycoprotein to induce cell fusion was not the result of an inhibition by cell-associated CD4, since fusion was also not observed when rVVST-infected CD4- cells were cocultured with CD4+ cells. Thus, the HIV-2ST envelope protein itself is defective in its ability to induce cell fusion. Furthermore, the expression, processing, transport, and surface stability of env products of HIV-2ST are unlikely to be responsible for its attenuation, suggesting that the molecular interactions between its env products and target cell membranes are significantly altered.
Assuntos
HIV-2/patogenicidade , Proteínas dos Retroviridae/fisiologia , Proteínas do Envelope Viral/fisiologia , Antígenos CD4 , Fusão Celular/genética , Fusão Celular/fisiologia , Linhagem Celular , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , Células Gigantes/patologia , HIV-2/genética , HIV-2/fisiologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/fisiologia , Proteínas dos Retroviridae/genética , Vaccinia virus/genética , Proteínas do Envelope Viral/genéticaRESUMO
We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/análise , Inibidores Enzimáticos/farmacologia , Isoenzimas/análise , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/genética , Agonistas Adrenérgicos beta/farmacologia , Animais , Células CHO , Células COS , Catálise , Cricetinae , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoproterenol/farmacologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Fatores de Tempo , TransfecçãoRESUMO
We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologia , Piridinas/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Ligação Competitiva , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Catálise/efeitos dos fármacos , AMP Cíclico/biossíntese , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Cobaias , Humanos , Masculino , Camundongos , Inibidores de Fosfodiesterase/química , Ligação Proteica/efeitos dos fármacos , Piridinas/química , Pirrolidinonas/antagonistas & inibidores , Pirrolidinonas/metabolismo , Rolipram , TrítioRESUMO
Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.
Assuntos
Anticorpos Monoclonais/química , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Imunoterapia , Glicoproteínas de Membrana/imunologia , Mucinas/imunologia , Neoplasias Ovarianas/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Neoplasias da Mama/terapia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Repetições Minissatélites , Dados de Sequência Molecular , Mucina-1 , Proteínas de Neoplasias/imunologia , Neoplasias Ovarianas/terapia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.
Assuntos
Enzimas/química , Enzimas/metabolismo , Rhabdoviridae/enzimologia , Rhabdoviridae/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Baculoviridae/genética , Pesquisa Biomédica/organização & administração , Pesquisa Biomédica/tendências , Enzimas/genética , Escherichia coli/genética , União Europeia , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição Gênica , Proteínas não Estruturais Virais/genéticaRESUMO
Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Algoritmos , Meios de Cultura , Vetores Genéticos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Solubilidade , TemperaturaRESUMO
Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.
Assuntos
Proteínas/metabolismo , Proteômica/métodos , Cristalização , Hidrólise , Luz , Espectrometria de Massas , Microscopia de Fluorescência , Modelos Moleculares , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espalhamento de Radiação , Tripsina , Ultracentrifugação , Raios XRESUMO
The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
Assuntos
Infecções Bacterianas/metabolismo , Proteínas de Bactérias/química , Proteômica/métodos , Proteínas Virais/química , Viroses/metabolismo , Animais , Infecções Bacterianas/microbiologia , Humanos , Dobramento de Proteína , Viroses/virologiaRESUMO
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
Assuntos
Bacillus anthracis/genética , Proteômica/métodos , Bacillus cereus/genética , Proteínas de Bactérias , Clonagem Molecular , Biologia Computacional , Cristalização , Cristalografia por Raios X , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Espectroscopia de Ressonância Magnética , RNA de Transferência/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Robótica , Esporos Bacterianos/genética , SulfurtransferasesRESUMO
The envelope glycoprotein of human immunodeficiency virus consists of two subunits, designated gp120 and gp41, derived from the cleavage of a precursor polypeptide gp160. When expressed from a recombinant vaccinia virus and analyzed by velocity gradient sedimentation and polyacrylamide gel electrophoresis, a significant proportion of gp160 molecules formed oligomers that were stabilized by intermolecular disulfide bonds. Oligomeric forms of both gp120 and gp41 were also observed, but these oligomers were noncovalently associated. Both the intermolecularly linked oligomers of gp160 and the unlinked oligomeric envelope protein subunits were found to accumulate with time. These results indicate that there are two populations of gp160 precursors, one that is folded and processed correctly into gp120 and gp41 and another that is intermolecularly disulfide bonded and remains uncleaved. We propose that the formation of intermolecular disulfide bonds is not an intermediate step in the maturation of the envelope glycoprotein, but rather a result of misfolding of the gp160 precursor which prevents it from being properly processed.