RESUMEN
A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Alérgenos/biosíntesis , Proteínas Portadoras/genética , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Proteínas de Plantas/biosíntesis , Alérgenos/aislamiento & purificación , Secuencia de Aminoácidos , Antígenos de Plantas , Secuencia de Bases , Proteínas Portadoras/química , División Celular/efectos de los fármacos , Clonación Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Polen/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Linfocitos T/efectos de los fármacosRESUMEN
Three isoforms of the major birch pollen allergen, Bet v, 1 from Betula verrucosa have been expressed as recombinant proteins in E. coli and purified. The immunochemical properties of recombinant isoforms (rBet v 1) differed on immunoblots when compared using Mabs and birch pollen allergic patients serum IgE. 2-D gel analysis showed that recombinant isoforms with different epitope structure can focus under the same protein spot after electrophoresis. The structure of conformational epitopes can be distorted by amino acid substitutions even when T-cell epitopes are not affected as judged by T-cell proliferation studies.
Asunto(s)
Alérgenos/inmunología , Isoenzimas/inmunología , Proteínas de Plantas/inmunología , Polen/inmunología , Alérgenos/genética , Secuencia de Aminoácidos , Antígenos de Plantas , Electroforesis en Gel Bidimensional , Isoenzimas/genética , Datos de Secuencia Molecular , Extractos Vegetales/inmunología , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , ÁrbolesRESUMEN
A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminoácidos Diaminos , Carboxipeptidasas/metabolismo , Leucina , Mutación Puntual , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Carboxipeptidasas/biosíntesis , Carboxipeptidasas/química , Catepsina A , Escherichia coli , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of L-configuration with basic amino acid residues in either P1 or P1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P1 could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.
Asunto(s)
Carboxipeptidasas/metabolismo , Penicillium/enzimología , Amidas/metabolismo , Secuencia de Aminoácidos , Carboxipeptidasas/aislamiento & purificación , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Análisis de Secuencia , Especificidad por SustratoRESUMEN
The possibilities of obtaining polypeptide amides in good yields from polypeptide acids by carboxypeptidase catalyzed transpeptidation have been investigated in different C-terminal amidations catalysed by CPD-Y, placing particular emphasis on the structure of the leaving group. In models for GRF(1-29) acid, larger hydrophilic leaving groups like threonine were particularly useful in reactions with arginine amide acting as nucleophile. In models for calcitonin acids, good results were obtained using also large hydrophobics like methionine and tryptophane in amidations with ammonia acting as nucleophile. Influences on both rate and synthetic efficiency of similar trend, but different magnitude, were observed in both salmon and human calcitonin models and high yields were attainable also in longer peptides or if recombinant secreted enzyme was employed.
Asunto(s)
Carboxipeptidasas/química , Péptidos/síntesis química , Amidas/síntesis química , Amidas/química , Secuencia de Aminoácidos , Calcitonina/síntesis química , Calcitonina/química , Modelos Químicos , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Six H-GluGlyArg-anilides with different ortho or para substituents in the aniline group, and H-GluGlyArg-benzylamide were synthesized. KM and kcat for the urokinase-catalyzed hydrolysis of the compounds were determined at 37 degrees C and pH 7.40. In the initial rate measurements, HPLC was used for product quantitation. KM varied between 0.20 mM and 8.9 mM, whereas kcat varied between 0.8s-1 and 16.5s-1 for the investigated substrates. A Hammett plot and a "Charton plot" of the rate data are presented. kcat were, to a minor extent, dependent on the pKa of the leaving group, whereas steric effects had a more marked influence on the overall rate constant. A o-benzyl substituent in the aniline-leaving group exerts less sterical hindrance to the enzymatic hydrolysis than expected from the Charton plot. The significance of the results in relation to the development of urokinase labile dextran prodrugs in discussed.