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2.
Proc Natl Acad Sci U S A ; 98(24): 13514-8, 2001 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-11707596

RESUMO

We have developed a methodology of prodrug delivery by using a modified insulin species whose biological activity potentially can be regulated in vivo. Native insulin was derivatized with aldol-terminated chemical modifications that can be selectively removed by the catalytic aldolase antibody 38C2 under physiologic conditions. The derivatized organoinsulin (insulin(D)) was defective with respect to receptor binding and stimulation of glucose transport. The affinity of insulin(D) for the insulin receptor was reduced by 90% in binding studies using intact cells. The ability of insulin(D) to stimulate glucose transport was reduced by 96% in 3T3-L1 adipocytes and by 55% in conscious rats. Incubation of insulin(D) with the catalytic aldolase antibody 38C2 cleaved all of the aldol-terminated modifications, restoring native insulin. Treatment of insulin(D) with 38C2 also restored insulin(D)'s receptor binding and glucose transport-stimulating activities in vitro, as well as its ability to lower glucose levels in animals in vivo. We propose that these results are the foundation for an in vivo regulated system of insulin activation using the prohormone insulin(D) and catalytic antibody 38C2 with potential therapeutic application.


Assuntos
Anticorpos Catalíticos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Insulina/metabolismo , Precursores de Proteínas/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Catálise , Linhagem Celular , Glucose/metabolismo , Humanos , Insulina/biossíntese , Masculino , Camundongos , Precursores de Proteínas/biossíntese , Ratos , Ratos Wistar , Receptor de Insulina/metabolismo
3.
J Mol Biol ; 314(1): 93-102, 2001 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-11724535

RESUMO

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.


Assuntos
Anticorpos Catalíticos/química , Anticorpos Catalíticos/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Anticorpos Catalíticos/imunologia , Sítios de Ligação de Anticorpos , Catálise , Cátions/metabolismo , Cristalografia por Raios X , Entropia , Haptenos/química , Haptenos/imunologia , Haptenos/metabolismo , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Conformação Proteica , Solventes , Eletricidade Estática , Relação Estrutura-Atividade
4.
Bioorg Med Chem Lett ; 11(22): 2983-6, 2001 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-11677141

RESUMO

We developed a visible detection system for antibody-catalyzed retro-aldol-retro-Michael reactions. Aldolase antibody 38C2 catalyzed the reaction of substrate 1 to provide 6-bromo-2-napthol that forms a visible colored azo dye with diazonium salts. This system has potential for the screening of novel catalysts.


Assuntos
Anticorpos Catalíticos/metabolismo , Frutose-Bifosfato Aldolase/imunologia , Animais , Bactérias/efeitos dos fármacos , Biotransformação , Catálise , Compostos de Diazônio , Hibridomas/efeitos dos fármacos , Cinética , Camundongos , Naftóis/química
5.
Science ; 293(5536): 1806-11, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11546867

RESUMO

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.


Assuntos
Anticorpos Catalíticos/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxidantes/metabolismo , Oxigênio/metabolismo , Água/química , Água/metabolismo , Animais , Anticorpos Catalíticos/química , Sítios de Ligação , Catálise , Sequência Conservada , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Oxidantes/química , Oxirredução , Conformação Proteica , Oxigênio Singlete , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica , Triptofano/metabolismo , Raios Ultravioleta , Xenônio/metabolismo
7.
Proc Natl Acad Sci U S A ; 98(13): 7528-33, 2001 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-11404472

RESUMO

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 10(2)-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.


Assuntos
Anticorpos Catalíticos/metabolismo , Etoposídeo/farmacocinética , Etoposídeo/toxicidade , Pró-Fármacos/farmacocinética , Pró-Fármacos/toxicidade , Animais , Biotransformação , Catálise , Divisão Celular/efeitos dos fármacos , Frutose-Bifosfato Aldolase/imunologia , Cinética , Camundongos , Estrutura Molecular , Neuroblastoma , Pró-Fármacos/síntese química , Fatores de Tempo , Células Tumorais Cultivadas
8.
Chemistry ; 7(8): 1691-702, 2001 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-11349910

RESUMO

Naturally occurring epothilones have been synthesized starting from enantiomerically pure aldol compounds 9-11, which were obtained by antibody catalysis. Aldolase antibody 38C2 catalyzed the resolution of (+/-)-9 by enantioselective retro-aldol reaction to afford 9 in 90% ee at 50 % conversion. Compounds 10 and 11 were obtained in more than 99% ee at 50% conversion by resolution of their racemic mixtures using newly developed aldolase antibodies 84G3, 85H6 or 93F3. Compounds 9, 10 and 11 were resolved in multigram quantities and then converted to the epothilones by metathesis processes, which were catalyzed by Grubbs' catalysts.


Assuntos
Antibacterianos/síntese química , Anticorpos Catalíticos/metabolismo , Antineoplásicos/síntese química , Epotilonas , Compostos de Epóxi/síntese química , Tiazóis/síntese química , Antibióticos Antineoplásicos/síntese química , Frutose-Bifosfato Aldolase/metabolismo , Haptenos/metabolismo , Macrolídeos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Myxococcales/metabolismo , Estereoisomerismo
9.
J Org Chem ; 66(5): 1725-32, 2001 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-11262119

RESUMO

The synthesis of the first examples of stilbene-tethered hydrophobic C-nucleosides is described. Compounds of this type are targeted for use with our recently reported "blue-fluorescent antibodies" with the aim of probing native and nonnatural DNA. The nucleophilic addition of aryl Grignard reagents to either a protected 2'-deoxy-1'-chloro-ribofuranose or a protected 2'-deoxy-ribonolactone was the key synthetic step and afforded C-nucleosides in good yields. Both routes resulted in a final product that was >/=90% of the beta-anomer. Amide- and ether-based linkers for attachment of trans-stilbene to the nucleobase were assessed for utility during synthesis and in binding of the ligands to a blue-fluorescent monoclonal antibody. X-ray structures of each complex were obtained and serve as a guideline for second-generation stilbene-tethered C-nucleosides. The development of these hydrophobic nucleosides will be useful in current native and nonnatural DNA studies and invaluable for investigations regarding novel, nonnatural genomes in the future.


Assuntos
Anticorpos/química , Nucleosídeos/síntese química , Estilbenos/química , Cristalografia por Raios X , Fluorescência , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular
10.
Chembiochem ; 2(9): 656-65, 2001 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-11828502

RESUMO

Three monoclonal aldolase antibodies (84G3, 85H6, and 93F3), generated against a beta-diketone hapten (II) by the reactive immunization technique, catalyzed highly enantioselective retro-aldol reactions of the racemic thiazole aldols 13-20. Antibody 84G3 (0.0004-0.005 mol%) was used to resolve (+/-)-13-(+/-)-18 to afford compounds 13-18 in multigram quantities. Multiple 13-alkyl analogues of epothilone (7-12) and their trans isomers ((E)-7-(E)-12) were synthesized starting from thiazole aldols 13-18. Construction of the trisubstituted olefin moiety in compounds 7-12 and (E)-7-(E)-12 was catalyzed by Grubbs' catalyst (X). Initial biological testing with compounds 7-10 and their trans isomers showed that compounds 9, 10, and (E)-10 have appreciable tubulin polymerization and antiproliferative activities that approached those of epothilone C. The most active compound, (E)-9, even displayed potencies comparable to those observed for epothilones A and D. Interestingly, all trans analogues were more potent than their corresponding cis isomers. While introduction of an alkyl group at C-13 in the cis series led to an overall reduction in biological activity (compared to epothilone C), appropriate modification of the thiazole moiety (replacement of the 2-methyl substituent by a 2-methylthio group) was able to compensate for this loss. These results are encouraging in view of the expectation that epoxidations of these compounds should further increase their cellular activities. Thus, compounds 9, 10, and (E)-9 and (E)-10 represent highly promising candidates for further studies.


Assuntos
Anticorpos/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Epotilonas , Macrolídeos/síntese química , Macrolídeos/farmacologia , Tiazóis/química , Aldeído Liases/química , Aldeído Liases/imunologia , Alquilação , Animais , Antineoplásicos/química , Catálise , Humanos , Lactonas/síntese química , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo , Suínos , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/efeitos dos fármacos , Células Tumorais Cultivadas
11.
Science ; 290(5490): 307-13, 2000 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-11030644

RESUMO

The forte of catalytic antibodies has resided in the control of the ground-state reaction coordinate. A principle and method are now described in which antibodies can direct the outcome of photophysical and photochemical events that take place on excited-state potential energy surfaces. The key component is a chemically reactive optical sensor that provides a direct report of the dynamic interplay between protein and ligand at the active site. To illustrate the concept, we used a trans-stilbene hapten to elicit a panel of monoclonal antibodies that displayed a range of fluorescent spectral behavior when bound to a trans-stilbene substrate. Several antibodies yielded a blue fluorescence indicative of an excited-state complex or "exciplex" between trans-stilbene and the antibody. The antibodies controlled the isomerization coordinate of trans-stilbene and dynamically coupled this manifold with an active-site residue. A step was taken toward the use of antibody-based photochemical sensors for diagnostic and clinical applications.


Assuntos
Anticorpos Catalíticos/química , Anticorpos Monoclonais/química , Fluorescência , Estilbenos/química , Estilbenos/imunologia , Sítios de Ligação , Sítios de Ligação de Anticorpos , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Haptenos , Ligantes , Microscopia de Fluorescência , Modelos Químicos , Modelos Moleculares , Fotoquímica , Espectrometria de Fluorescência , Estereoisomerismo , Temperatura , Raios Ultravioleta
12.
Proc Natl Acad Sci U S A ; 97(20): 10930-5, 2000 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-11005865

RESUMO

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody-antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.


Assuntos
Anticorpos/química , Reações Antígeno-Anticorpo , Antígenos/química , Animais , Anticorpos/imunologia , Antígenos/imunologia , Catálise , Cricetinae , Humanos , Peróxido de Hidrogênio/química , Camundongos , Oxigênio/química , Ovinos
13.
Chemistry ; 6(15): 2772-4, 2000 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-10985725

RESUMO

The preparative scale kinetic resolution of racemic aldols 1-4 using aldolase antibodies 38C2 (Aldrich no. 47995-0) and 84G3 (Aldrich no. 52785-8) is described. These reactions use a biphasic aqueous/organic solvent system that allows the catalyst to be reused. Reaction scales range from miligrams to grams, with 0.0086 to 0.12 mol% of antibody binding sites. Because antibodies 38C2 and 84G3 have opposite enantioselectivities, both aldol product enantiomers are accessible by kinetic resolution.


Assuntos
Aldeídos/isolamento & purificação , Anticorpos Catalíticos , Frutose-Bifosfato Aldolase , Fragmentos Fab das Imunoglobulinas , Aldeídos/química , Indicadores e Reagentes , Cinética , Estereoisomerismo
14.
Bioorg Med Chem ; 8(5): 995-1003, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10882011

RESUMO

The syntheses of a class of three haptens derived from the same 4-aza-steroidal skeleton is described. The sequence begins with oxidative cleavage of ring A of commercially available, optically pure lithocholic acid. Insertion of nitrogen at position 4 and stereoselective hydrogenation of the resulting electron-rich enelactam under 600 psi H2 yielded a system analogous to testosterone-5-alpha-reductase inhibitors. Upon exhaustive reduction of this compound with lithium aluminium hydride, a linker for bioconjugation was attached before the N-oxide key functionality is established in ring A. This functional group is believed to be a true transition-state mimic for the electronic nature of initiation of the cationic cyclization of 2,3-epoxy-squalene derivatives. In addition, it also holds promise for eliciting acidic residues as part of a bait-and-switch strategy. Remarkably, both N-oxide epimers obtained from mCPBA oxidation can be separated by column chromatography on a 60 mg scale and were used in enantiopure form for separate immunizations. Reliable configurative assignment was carried out by comparison studies with previously characterized and published systems. A catalytic antibody (HA8-25A10) was obtained from the immunization with the hapten bearing an aminoxide oxygen in the beta position. Surprisingly, an inhibition study showed that the isomer with the inverted configuration at the N-oxide bound more strongly to this catalytic antibody.


Assuntos
Anticorpos Catalíticos/química , Compostos Aza/química , Haptenos/química , Ácido Litocólico/análogos & derivados , Oxirredutases/metabolismo , Anticorpos Catalíticos/metabolismo , Ácido Litocólico/química , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
15.
Proc Natl Acad Sci U S A ; 97(8): 3878-83, 2000 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-10760259

RESUMO

Catalytic aldolase antibodies generated by immunization with two different, but structurally related, beta-diketone haptens were cloned and sequenced to study similarities and differences between independently evolved catalysts. Kinetic and sequence analysis coupled with mutagenesis, structural, and modeling studies reveal that the defining event in the evolution of these catalysts was a somatic mutation that placed a lysine residue in a deep, yet otherwise unrefined, hydrophobic pocket. We suggest that covalent chemistries may be as readily selected from the immune repertoire as the traditional noncovalent interactions that have formed the basis of immunochemistry until this time. Further, we believe that these experiments recapitulate the defining events in the evolution of nature's enzymes, particularly as they relate to chemical mechanism, catalytic promiscuity, and gene duplication.


Assuntos
Anticorpos Catalíticos/metabolismo , Evolução Molecular , Sequência de Aminoácidos , Animais , Anticorpos Catalíticos/administração & dosagem , Anticorpos Catalíticos/genética , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Frutose-Bifosfato Aldolase/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Homologia de Sequência de Aminoácidos
16.
Science ; 287(5462): 2486-92, 2000 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-10741968

RESUMO

Messenger RNA levels were measured in actively dividing fibroblasts isolated from young, middle-age, and old-age humans and humans with progeria, a rare genetic disorder characterized by accelerated aging. Genes whose expression is associated with age-related phenotypes and diseases were identified. The data also suggest that an underlying mechanism of the aging process involves increasing errors in the mitotic machinery of dividing cells in the postreproductive stage of life. We propose that this dysfunction leads to chromosomal pathologies that result in misregulation of genes involved in the aging process.


Assuntos
Envelhecimento/genética , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Mitose , Progéria/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Fenômenos Bioquímicos , Divisão Celular , Linhagem Celular , Núcleo Celular/ultraestrutura , Criança , Segregação de Cromossomos/genética , Doença/etiologia , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Mitose/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Progéria/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/metabolismo , Fatores de Transcrição/genética
17.
Biochemistry ; 38(22): 7062-74, 1999 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10353817

RESUMO

The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.


Assuntos
Anticorpos Catalíticos/química , Sítios de Ligação de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos Catalíticos/biossíntese , Anticorpos Catalíticos/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Catálise , Cristalografia por Raios X , Haptenos/química , Haptenos/imunologia , Hidrolases/química , Hidrolases/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 96(12): 6925-30, 1999 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-10359815

RESUMO

Chemotherapeutic regimes are typically limited by nonspecific toxicity. To address this problem we have developed a broadly applicable drug-masking chemistry that operates in conjunction with a unique broad-scope catalytic antibody. This masking chemistry is applicable to a wide range of drugs because it is compatible with virtually any heteroatom. We demonstrate that generic drug-masking groups may be selectively removed by sequential retro-aldol-retro-Michael reactions catalyzed by antibody 38C2. This reaction cascade is not catalyzed by any known natural enzyme. Application of this masking chemistry to the anticancer drugs doxorubicin and camptothecin produced prodrugs with substantially reduced toxicity. These prodrugs are selectively unmasked by the catalytic antibody when it is applied at therapeutically relevant concentrations. We have demonstrated the efficacy of this approach by using human colon and prostate cancer cell lines. The antibody demonstrated a long in vivo half-life after administration to mice. Based on these findings, we believe that the system described here has the potential to become a key tool in selective chemotherapeutic strategies.


Assuntos
Anticorpos/administração & dosagem , Sistemas de Liberação de Medicamentos , Pró-Fármacos , Animais , Anticorpos/toxicidade , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Camptotecina/administração & dosagem , Camptotecina/toxicidade , Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Desenho de Fármacos , Humanos , Camundongos , Pró-Fármacos/administração & dosagem , Pró-Fármacos/toxicidade , Células Tumorais Cultivadas
20.
Proc Natl Acad Sci U S A ; 96(11): 6025-30, 1999 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-10339535

RESUMO

The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy- and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.


Assuntos
Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/biossíntese , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Bacteriófagos/genética , Sequência de Bases , Catálise , Primers do DNA , Dimerização , Epitopos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/ultraestrutura , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/ultraestrutura , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/ultraestrutura , Camundongos , Microscopia Eletrônica , Oligopeptídeos , Peptídeos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Proteínas Virais/biossíntese , Proteínas Virais/genética
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