RESUMEN
Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.
Asunto(s)
Cisteína/análogos & derivados , Inmunotoxinas/química , Proteínas de Plantas/química , Ribosomas/efectos de los fármacos , Toxinas Biológicas , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cisteína/química , Cisteína/inmunología , Pruebas Inmunológicas de Citotoxicidad , Disulfuros/química , Estabilidad de Medicamentos , Escherichia coli/metabolismo , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas , Proteínas de Neoplasias/biosíntesis , Proteínas de Plantas/inmunología , Proteínas de Plantas/farmacología , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Cecropin A is a naturally occurring peptide with bactericidal activity against gram-negative and gram-positive bacteria. Production of large quantities of bactericidal peptides that are similar in structure and activity to cecropin A has been achieved by combining recombinant DNA techniques and techniques and chemical modification. Expression of the bactericidal peptide in Escherichia coli was accomplished through the formation of a fusion protein. The 5' end of the L-ribulokinase gene was fused to a single copy of a synthetic gene encoding cecropin A. A methionine codon was engineered between the two genes, and a methionylglycine extension was introduced at the C terminus of cecropin A. Cyanogen bromide treatment of the fusion protein yielded cecropin A with a C-terminal homoserine. The recombinant cecropin A with a homoserine at the C terminus did not kill most gram-positive bacteria tested. However, recombinant cecropin A with a chemically modified C terminus has antimicrobial activity similar to that of cecropin produced by cecropia pupae.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos , Hormonas de Insectos/farmacología , Péptidos/farmacología , Antibacterianos/biosíntesis , ADN Recombinante/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etilenodiaminas/farmacología , Bacterias Grampositivas/efectos de los fármacos , Hormonas de Insectos/biosíntesis , Hormonas de Insectos/genética , Pruebas de Sensibilidad Microbiana , Biosíntesis de Péptidos , Péptidos/genética , Plásmidos/genética , Relación Estructura-ActividadRESUMEN
We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.
Asunto(s)
Antígenos CD/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunotoxinas/metabolismo , Ricina/metabolismo , Secuencia de Aminoácidos , Reacciones Antígeno-Anticuerpo , Secuencia de Bases , Unión Competitiva , Antígenos CD5 , Línea Celular , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Ingeniería Genética , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Inmunotoxinas/toxicidad , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/efectos de los fármacosRESUMEN
The peh gene, encoding polygalacturonase (Peh), was identified in Erwinia carotovora strain EC and cloned in Escherichia coli. Recombinant Peh (re-Peh) was purified from E. coli strain 706 containing peh on a recombinant plasmid. The activity of the re-Peh protein is optimal at pH 5.5. The N-terminal and internal amino acid (aa) sequences of re-Peh were determined and compared to the aa sequence deduced from the nucleotide (nt) sequence of the cloned peh. The re-Peh has no similarity, based on either the nt sequences or the deduced aa sequences, to pectate lyases from the same Er. carotovora strain or other organisms.
Asunto(s)
Pectobacterium carotovorum/genética , Poligalacturonasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Pectobacterium carotovorum/enzimología , Plásmidos , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Biosíntesis de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.
Asunto(s)
Alérgenos , Antibióticos Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Proteínas Fúngicas/farmacología , Genes Sintéticos , Inmunotoxinas/toxicidad , Ribonucleasas , Linfocitos T/efectos de los fármacos , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos de Plantas , Aspergillus/genética , Secuencia de Bases , Línea Celular , Células Cultivadas , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Globinas/genética , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Conejos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Mapeo Restrictivo , Reticulocitos/metabolismo , Linfocitos T/citologíaRESUMEN
A practical program edited by BASIC was used in this test. The test included 7 indexes of fluid intelligence by using the method of talking between man and computer: speed of mental mathematic, digit-symbol, choice reaction time, counting visual number span, tracing reaction and recognition of meaningless figures. Through the principal component analysis and multiple stepwise regression techniques, the authors found out the mathematical model of the intelligence aging from 506 normal subjects and established a measuring system for the declined fluid intelligence and physiological age could be measured with the system. The correlation of the repetition of the test was 0.9528. This method was suitable for mental workers of 46-75 years old. The measured accuracy among the subjects of 50-70 years old was higher. The method was applied practically in clinical evaluation of the effects of traditional herbal medicines and Qigong on fluid intelligence with aging. Through the analysis of validity and reliability, it was proved that this method was suitable for geriatric research.
Asunto(s)
Envejecimiento/psicología , Pruebas de Inteligencia , Medicina Tradicional China , Factores de Edad , Anciano , Femenino , Humanos , Masculino , Microcomputadores , Persona de Mediana EdadRESUMEN
The pelA gene from Erwinia carotovora strain EC encodes pectate lyase A (PLa). The gene was cloned and sequenced. The PLa was purified from Escherichia coli containing the pelA gene on a recombinant plasmid. The optimum pH for the enzyme reaction is at pH 8.5; the optimum temperature for the enzyme reaction is at 40 degrees C; and the purified PLa alone can macerate potato slices. Nucleotide sequence of pelA and the deduced polypeptide sequence of PLa were compared with the pelB gene of E. carotovora and the deduced amino acid sequence of PLb. The nucleotide sequences are 82% homologous and the amino acid sequences are 88% homologous.
Asunto(s)
Erwinia/genética , Proteínas de Plantas/genética , Polisacárido Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Genes , Datos de Secuencia MolecularRESUMEN
The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.
Asunto(s)
ADN Bacteriano/análisis , Erwinia/genética , Genes Bacterianos , Polisacárido Liasas/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Erwinia/enzimología , Regulación de la Expresión Génica , Fragmentos de Péptidos , Polisacárido Liasas/análisis , Polisacárido Liasas/metabolismo , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido NucleicoRESUMEN
Polygalacturonase (PG) was purified from Erwinia carotovora EC. A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E. carotovora genomic library by an immunological screening method. A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices. In addition, the E. coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices. Our results suggest that PG plays an important role in soft-rot disease.
Asunto(s)
Erwinia/enzimología , Glicósido Hidrolasas/metabolismo , Enfermedades de las Plantas , Plantas/metabolismo , Poligalacturonasa/metabolismo , Clonación Molecular , Enzimas de Restricción del ADN , Desoxirribonucleasa HindIII , Escherichia coli/genética , Genes , Genes Bacterianos , Poligalacturonasa/genética , Poligalacturonasa/aislamiento & purificaciónRESUMEN
The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.
Asunto(s)
Arabinosa/metabolismo , Proteínas Bacterianas , Erwinia/genética , Genes Bacterianos , Genes Reguladores , Proteínas Represoras/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Factor de Transcripción de AraC , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Erwinia/metabolismo , Proteínas de Escherichia coli , Prueba de Complementación Genética , Mutación , Plásmidos , Salmonella typhimurium/genética , Transcripción GenéticaRESUMEN
A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.
Asunto(s)
ADN , Bacteriófagos/genética , Secuencia de Bases , Precipitación Química , Clonación Molecular , Enzimas de Restricción del ADN , Desoxirribonucleasa I , Erwinia/genética , Escherichia coli/genética , Genes Reguladores , TransfecciónRESUMEN
Hybrid plasmids containing the araBAD operon of Salmonella typhimurium LT2 were characterized by Southern blot and genetic analyses. The nucleotide sequence of araB was determined. The araB gene product, ribulokinase (EC 2.7.1.16), was purified and the results of amino acid composition analysis and partial amino acid sequence are in agreement with predictions from the DNA sequence. Ribulokinase is 569 amino acid residues long and has a calculated Mr of 61 793. Ribulokinase shares significant homology with xylulose kinase from Escherichia coli. Codon usage in the araB gene does not favor those codons which have intermediate codon-anticodon binding energy.
Asunto(s)
Proteínas Bacterianas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol) , Fosfotransferasas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Codón/genética , Escherichia coli/genética , Genes , Genes Bacterianos , Operón , Fosfotransferasas/aislamiento & purificación , Especificidad de la EspecieRESUMEN
The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.
Asunto(s)
Isomerasas Aldosa-Cetosa , Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Carbohidrato Epimerasas/aislamiento & purificación , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Genes , Genes Bacterianos , OperónRESUMEN
The araD gene of Salmonella typhimurium LT2 consists of 744 nucleotides and has an ORF coding for a protein of 248 amino acid residues with a calculated Mr of 27059. The product of araD, L-ribulose-5-phosphate 4-epimerase, was purified and the amino terminal sequence was determined. It is identical to that predicted by the ORF. A 143-bp intercistronic region was found between the araA and araD genes which can form stem-loop structures with calculated free energies of up to -76.7 kcal/mol. Three sets of sequences within this 143-bp region are comparable to a consensus sequence deduced from several known intercistronic regions. Approximately equimolar amounts of the araBAD operon products were present in L-arabinose-induced cells. Sequences similar to both rho-independent and rho-dependent transcription terminators are present after the araD gene.
Asunto(s)
Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Carbohidrato Epimerasas/aislamiento & purificación , Clonación Molecular , Codón/genética , Escherichia coli/genética , Conformación de Ácido Nucleico , Operón , Regiones Terminadoras Genéticas , Transcripción GenéticaRESUMEN
A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method. A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E. carotovora culture supernatants. The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE. None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space. Plant tissue was macerated by the PLs made in E. coli.