RESUMO
Beaded cellulose (Perloza) was modified with acrylonitrile followed by reduction with diborane to give a functionalised support containing aminopropyl groups. This spacer arm was then further extended with a glycolamido or an Fmoc-amino acid-4-oxymethylphenoxyacetyl moiety. A number of peptides, including the Merrifield test peptide leucyl-alanyl-glycyl-valine, leucine-enkephalin, Acyl Carrier Protein (65-74), angiotensin I and II, ACTH(4-11) and LHRH were synthesised on the aminopropyl beaded cellulose support using modified t-butyloxycarbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) synthesis protocols. The peptides were cleaved from the support and further purified.
Assuntos
Celulose , Peptídeos/síntese química , Sequência de Aminoácidos , Aminoácidos/química , Dimetilformamida/química , Dioxanos/química , Fluorenos/química , Ésteres do Ácido Fórmico/química , Dados de Sequência Molecular , Oligopeptídeos/química , Tamanho da PartículaRESUMO
Aminopropyl Perloza beaded cellulose was used as the support for solid-phase synthesis of resin-bound Val-dLeu-Pro-Phe-Phe-Val-dLeu, an inhibitor of aspartic proteases. Both Boc and Fmoc SPPS methodologies were employed in separate syntheses. The peptide-resins were characterized by amino acid analysis. The peptide-resin from the Fmoc synthesis gave the better amino acid analysis of the two syntheses and was used for further studies. Following modification of the peptide N-terminus by succinylation, the peptide-resin was able to bind chymosin (E.C. 3.5.21.4). The peptide resin was used for isolation of chymosin from a crude recombinant broth.
Assuntos
Cromatografia de Afinidade , Quimosina/isolamento & purificação , Oligopeptídeos/síntese química , Inibidores de Proteases/síntese química , Sequência de Aminoácidos , Aminoácidos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Celulose , Quimosina/metabolismo , Fluorenos , Ésteres do Ácido Fórmico , Ligantes , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Inibidores de Proteases/química , Proteínas Recombinantes/isolamento & purificação , Resinas VegetaisRESUMO
Peptide-Perloza beaded cellulose conjugates were synthesized. However, the concentrated TFA solutions usually used for deprotecting peptide-support conjugates were found to be unsuitable for use with Perloza because they destroyed the chromatographic flow properties of the matrix. Conditions for non-destructive (to the matrix) deprotection of the resin-bound peptide were determined in this study. A cocktail of m-cesol/EDT/thioanisole/TMSBr/TFA/DCM (1:1:2:2:15:79 by volume-Reagent D) was found to cleave amino acid side-chain-protecting groups while leaving the chromatographic properties of the peptide-resin unaltered. Although treating aminopropyl Perloza with solutions of TFA in DCM resulted in decrease of amine substitution, treating peptide-Perloza conjugates with identical reagents gave minimal loss of peptide.
Assuntos
Oligopeptídeos/síntese química , Sequência de Aminoácidos , Celulose/química , Cromatografia/métodos , Dados de Sequência MolecularRESUMO
Perloza beaded cellulose was functionalised by a cyanoethylation/reduction procedure to give aminopropyl Perloza. Fmoc-amino acids were anchored to aminopropyl Perloza beaded cellulose via the TFA labile 4-oxymethylphenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxyacetyl-2,4-dichloro-phenyl esters, all 20 amino acids were anchored at substitution levels ranging from 0.37 to 0.65 mmol/g. Fmoc-amino acids were also anchored using the peptide-amide linker 4-[(R,S)-1-[1-(9H-fluoren-9-yl)-methoxycarbonylamino - (2',4'-dimethoxybenzyl]phenoxyacetic acid. The Fmoc-aminoacyl resins were used for SPPS using Fmoc chemistry. SPPS was carried out using either an LKB Biolynx 4175 low-pressure pumped column continuous-flow peptide synthesiser or an ABI 430A automated vortexing batchwise instrument. Comparison of peptides made using each synthesiser showed little difference in quality of the crude peptides. Different Fmoc-amino acid activation methods (DIC/HOBt/DMF, HBTU, DIC/HOBt/DCM) were found to be equally useful with Perloza. Peptides were cleaved using TFA plus scavengers; however, the TFA-swollen resin was not readily separated from the TFA/peptide solution by simple filtration. Therefore alternative cleavage workup procedures were used with Perloza. Peptides were purified by HPLC and characterised by HPLC and amino acid analysis, and in some cases by FAB-MS. Successful syntheses ranged from 5 to 34 amino acids in length. Some of the peptides were also synthesized using a polystyrene support and standardised (ABI Fastmoc) SPPS protocols. The crude cleaved peptides from each synthesis were compared by HPLC analysis. The overall aim of our work with Perloza is synthesis of resin-bound peptide ligands for affinity chromatography and antibody generation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aminoácidos , Celulose , Fluorenos , Peptídeos/síntese química , Sequência de Aminoácidos , Anticorpos/química , Cromatografia de Afinidade/métodos , Hempa , Dados de Sequência Molecular , Resinas VegetaisRESUMO
Aminopropyl derivatized Perloza beaded cellulose was acylated with alpha-bromoacetic anhydride to give alpha-bromo-acetamidopropyl Perloza. (N-Acetyl)-Cys-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the 7 C-terminal amino acids of the decapeptide luteinizing hormone-releasing hormone with a cysteine added to the N-terminus, was synthesized using Fmoc chemistry. The purified peptide (1.35-1.9 eq) was coupled to alpha-bromoacetamidopropyl Perloza in 0.1 M NaHCO3 solution, pH 8.3, for 1-2 hours. The peptide was anchored to the support via a thioether linkage. Analysis of the peptide-Perloza conjugate indicated near-quantitative displacement of support-bound bromine by the peptide. The peptidic affinity matrix was able to bind ovine antibodies to luteinizing hormone-releasing hormone (LHRH). Thioether immobilization offers directed, chemically stable, high-yield anchoring of synthetic peptides onto a chromatographic support. The high reaction efficiency means there is little waste of valuable synthetic peptide.
Assuntos
Celulose , Ligantes , Microesferas , Peptídeos/química , Acetatos/química , Acilação , Sequência de Aminoácidos , Anticorpos/metabolismo , Bromo/metabolismo , Eletroforese em Gel de Poliacrilamida , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , SulfetosRESUMO
Formation of a macromolecular complex between the RNA binding protein HIV-1 Rev and HIV-1 mRNA is an essential prerequisite for nuclear export and subsequent expression of HIV-1 mRNA. The arginine rich peptide TRQARRNRRRRWRARQR, corresponding to residues 34-50 of HIV-1 Rev, contains the mRNA binding motif. We prepared a thioether linked Rev34-50-cellulose conjugate to affinity purify a fragment of synthetic mRNA corresponding to the high affinity binding site for Rev. The correctly folded fraction of mRNA (27.5%) was isolated from a crude synthetic mixture.
Assuntos
Produtos do Gene rev/metabolismo , HIV-1/genética , Conformação de Ácido Nucleico , RNA Mensageiro/isolamento & purificação , RNA Viral/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia de Afinidade/métodos , Ligantes , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Desnaturação Proteica , RNA Mensageiro/química , RNA Viral/química , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
NMR spectroscopy has been used to solve the three-dimensional solution structure of a minimal RNA-binding domain of the Rev protein from the human immunodeficiency virus (type 1), an essential regulatory protein for viral replication. The presence of 10 arginine residues in the 17-residue peptide Rev34-50 caused significant problems in assignment of the NMR spectra. To improve spectral resolution, the peptide was synthesized with an alanine replacing a nonessential arginine and with selectively 15N-labeled residues. Contrary to Chou-Fasman modeling predictions an alpha-helix was detected in both water and 20% trifluoroethanol (TFE) and was found to span residues that constitute the RNA-binding and nuclear-localizing domains of Rev. The sequence-specific information provided by the NMR data gives a full description of the solution conformation of Rev34-50 which serves as a template for investigating binding of the peptide to RNA from the Rev response element (RRE). Preliminary modeling suggests that the helix can fit neatly into the expanded major groove of the RRE where interactions between the peptide side chains and the RNA can be identified. These data may aid the construction of a suitable pharmacophore model for the rational design of molecules that block Rev-RNA binding and inhibit HIV replication.
Assuntos
Produtos do Gene rev/química , HIV-1/metabolismo , Conformação Proteica , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética/métodos , Modelos Estruturais , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Peptídeos/síntese química , Peptídeos/química , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
In vivo and in vitro T cell responses to overlapping 20-mer peptides that span the entire 19-kDa protein of Mycobacterium tuberculosis have been compared in three different strains of mice. Immunization of the mice with peptides and analysis of specific antibody production is an in vivo assay of Th cell activity. Peptides 1-20 and 61-80 elicited strong IgG1 responses in BALB/cJ, C57BL/10J, and B10.BR mice, indicating that these peptides could stimulate Th cells, possibly of a Th2 phenotype. T cells isolated from peptide-immunized mice were challenged in vitro with peptide, and their proliferative responses were analyzed. T cells from these three strains of mice immunized with peptides 1-20, 61-80, and 76-95 also responded to challenge with specific peptide in vitro. In addition, B10.BR mice and BALB/cJ mice showed antibody and T cell proliferative responses to peptides 136-155 and 145-159, respectively. Thus, in vitro proliferating T cells were found to possess specificities for peptide epitopes that were almost identical to those of the antibody-producing cells. Delayed-type hypersensitivity (DTH) responses to these peptides were also examined in the three strains. Interestingly, the T cells responding in the DTH assay had Ag specificities that were quite different from those identified in the antibody and proliferation assays. These results suggested that DTH Th cells form a separate population from antibody Th and proliferative T cells and these populations of cells were differentially activated, in an Ag-specific manner.