RESUMO
Targeted zinc-finger (ZF) DNA-binding domains in conjunction with nuclear receptor ligand-binding domains (LBDs) produce chemically inducible gene switches that have applications in gene therapy and proteomic and genomic research. The benzoate X receptor-ß (BXRß) LBD was used to construct homodimer and single-chain ZF transcription factors (ZF(TF)s). These ZF(TF)s specifically regulated the transcription of target genes in response to two ligands, ethyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, in a dose-dependent manner. The ZF(TF)s also regulated the expression of endogenous intercellular adhesion molecule-1 in response to either ligand. The advantage of BXRß-based ZF(TF)s is that the ligands are inexpensive and easily synthetically modified, making the system a base for creation of orthogonal ligand-receptor pairs and expanding the gene-switch toolbox.
Assuntos
Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/genética , Proteínas de Xenopus/genética , Dedos de Zinco/genética , Técnicas Genéticas , Receptores Citoplasmáticos e Nucleares/química , Fatores de Transcrição/química , Proteínas de Xenopus/químicaRESUMO
Chemically inducible gene switches that regulate expression of endogenous genes have multiple applications for basic gene expression research and gene therapy. Single-chain zinc-finger transcription factors that utilize either estrogen receptor homodimers or retinoid X receptor-alpha/ecdysone receptor heterodimers are shown here to be effective regulators of ICAM-1 and ErbB-2 transcription. Using activator (VP64) and repressor (Krüppel-associated box) domains to impart regulatory directionality, ICAM-1 was activated by 4.8-fold and repressed by 81% with the estrogen receptor-inducible transcription factors. ErbB-2 was activated by up to threefold and repressed by 84% with the retinoid X receptor-alpha/ecdysone receptor-inducible transcription factors. The dynamic range of these proteins was similar to the constitutive system and showed negligible basal regulation when ligand was not present. We have also demonstrated that the regulation imposed by these inducible transcription factors is dose dependent, sustainable for at least 11 days and reversible upon cessation of drug treatment. Importantly, these proteins can be used in conjunction with each other with no detectable overlap of activity enabling concurrent and temporal regulation of multiple genes within the same cell. Thus, these chemically inducible transcription factors are valuable tools for spatiotemporal control of gene expression that should prove valuable for research and gene therapy applications.
Assuntos
Regulação da Expressão Gênica , Terapia Genética/métodos , Receptores de Superfície Celular/genética , Fatores de Transcrição/genética , Dedos de Zinco , Western Blotting/métodos , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Expressão Gênica/efeitos dos fármacos , Genes Reguladores , Genes erbB-2 , Engenharia Genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4+ T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4+ T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4+ T cells are refractory against this highly efficient primary route of infection. CD34+ cells transduced with the CAD-R5 vector gave rise to CD4+ and CD8+ thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4+ T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4+ T cells and CD34+ cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.
Assuntos
Anticorpos/genética , Linfócitos T CD4-Positivos/imunologia , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/fisiologia , Receptores CCR5/genética , Animais , Anticorpos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Células Cultivadas , Citoproteção , Células Dendríticas/imunologia , Células Dendríticas/virologia , Regulação da Expressão Gênica , Infecções por HIV/imunologia , Humanos , Camundongos , Camundongos SCID , Receptores CCR5/metabolismoRESUMO
BACKGROUND: Intracellular antibodies (intrabodies) have been used for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. Intrabodies present an alternative to methods of gene inactivation that target genomic DNA or m-RNA, such as RNA interference. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects in tumor growth. METHODS: In order to generate a precise tool for the simultaneous silencing of two independent signaling pathways essential for angiogenesis, we developed a bispecific, tetravalent endoplasmatic reticulum (ER)-targeted intradiabody, against Tie-2 and VEGF-R2. RESULTS: Using an adenovirus mediated gene delivery system, we achieved the simultaneous downregulation of the two cell surface receptors and demonstrate that the intradiabody is significantly more powerful with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2 when compared to scFv intrabodies. In a human melanoma xenograft mouse model, we could show that blockade of both VEGF-R2 and Tie-2 pathways or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2 % and 74.4 %). CONCLUSION: We demonstrate for the first time that simultaneous inhibition of the VEGF and the Tie-2 receptor pathways result in additive antiangiogenic effects in vitro and in vivo as compared to single VEGF receptor pathway blockade, strengthening the potential of simultaneous targeting of multiple pathways as a therapeutic strategy.
Assuntos
Inativação Gênica , Neoplasias/irrigação sanguínea , Neovascularização Patológica/genética , Fenótipo , Receptor TIE-2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Criança , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Melanoma Experimental/genética , Camundongos , Camundongos Nus , Transdução de Sinais/genética , Transplante HeterólogoRESUMO
[reaction--see text] Direct catalytic enantio- and diastereoselective Michael addition reactions of unmodified aldehydes to nitro olefins using (S)-2-(morpholinomethyl)pyrrolidine as a catalyst are described. The reactions proceed in good yield (up to 96%) in a highly syn-selective manner (up to 98:2) with enantioselectivities approaching 80%. The resulting gamma-formyl nitro compounds are readily converted to chiral, nonracemic 3,4-disubstituted pyrrolidines.
Assuntos
Aldeídos/química , Alcenos/química , Morfolinas/síntese química , Pirróis/síntese química , Pirrolidinas/síntese química , Catálise , Indicadores e Reagentes , Cinética , Estereoisomerismo , TemperaturaRESUMO
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/metabolismoRESUMO
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/químicaRESUMO
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 CultivadasRESUMO
Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.
Assuntos
HIV-1/química , Peptídeos/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por HIV/prevenção & controle , Humanos , Imunoglobulina G/imunologia , Testes de Neutralização , Peptídeos/química , Ligação Proteica , Sensibilidade e Especificidade , Proteínas do Envelope Viral/químicaRESUMO
In previous studies we have developed Cys(2)-His(2) zinc finger domains that specifically recognized each of the 16 5'-GNN-3' DNA target sequences and could be used to assemble six-finger proteins that bind 18-base pair DNA sequences (Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1495--1500). Such proteins provide the basis for the construction of artificial transcription factors to study gene/function relationships in the post-genomic era. Central to the universal application of this approach is the development of zinc finger domains that specifically recognize each of the 64 possible DNA triplets. Here we describe the construction of a novel phage display library that enables the selection of zinc finger domains recognizing the 5'-ANN-3' family of DNA sequences. Library selections provided domains that in most cases showed binding specificity for the 3-base pair target site that they were selected to bind. These zinc finger domains were used to construct 6-finger proteins that specifically bound their 18-base pair target site with affinities in the pm to low nm range. When fused to regulatory domains, these proteins containing various numbers of 5'-ANN-3' domains were capable of specific transcriptional regulation of a reporter gene and the endogenous human ERBB-2 and ERBB-3 genes. These results suggest that modular DNA recognition by zinc finger domains is not limited to the 5'-GNN-3' family of DNA sequences and can be extended to the 5'-ANN-3' family. The domains characterized in this work provide for the rapid construction of artificial transcription factors, thereby greatly increasing the number of sequences and genes that can be targeted by DNA-binding proteins built from pre-defined zinc finger domains.
Assuntos
DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Genes erbB-2 , Genes erbB , Biblioteca de Peptídeos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Simulação por Computador , DNA/genética , Primers do DNA , Receptores ErbB/genética , Genes Reporter , Genes erbB-1 , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Conformação Proteica , Estrutura Secundária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Fatores de Transcrição/síntese química , Fatores de Transcrição/químicaRESUMO
An intact human immunoglobulin with a full-length hinge has been crystallized for the first time in a form in which all of the Ig domains are ordered. The IgG1 antibody b12 is one of only three known monoclonal antibodies described that potently neutralize a broad range of HIV-1 primary isolates. It binds to an epitope overlapping the conserved CD4 binding site on the viral surface antigen gp120. Hexagonal crystals corresponding to space group R32 were grown from 0.8 M ammonium sulfate, with unit-cell parameters a = b = 271.3, c = 175.2 A and one molecule per asymmetric unit. The crystals diffract to 2.8 A and a preliminary molecular-replacement solution indicates that all 12 Ig domains of the antibody can be resolved.
Assuntos
HIV-1/imunologia , Imunoglobulinas/química , Cristalização , Infecções por HIV/imunologia , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/isolamento & purificação , Masculino , Testes de Neutralização , Conformação ProteicaRESUMO
Artificial transcription factors based on modified zinc-finger DNA-binding domains have been shown to activate or repress the transcription of endogenous genes in multiple organisms. Advances in both the construction of novel zinc-finger proteins and our understanding of the characteristics of a productive regulatory site have fueled these achievements.
Assuntos
Proteínas de Ligação a DNA/química , Sítios de Ligação , Biblioteca Gênica , Genoma , Genoma Humano , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Dedos de ZincoRESUMO
In order to construct zinc finger domains that recognize all of the possible 64 DNA triplets, it is necessary to understand the mechanisms of protein/DNA interactions on the molecular level. Previously we reported 16 zinc finger domains which had been characterized in detail to bind specifically to the 5'-GNN-3' family of DNA sequences. Artificial transcription factors constructed from these domains can regulate the expression of endogenous genes. These domains were created by phage-display selection followed by site-directed mutagenesis. A total of 84 mutants of a three-domain zinc finger protein have been analyzed for their DNA-binding specificity. Here, we report the results of this systematic and extensive mutagenesis study. New insights into zinc finger/DNA interactions were obtained by combining specificity data with computer modeling and comparison with known structural data from NMR and crystallographic studies. This analysis suggests that unusual cross-strand and inter-helical contacts are made by some of these proteins, and the general orientation of the recognition helix to the DNA is flexible, even when constrained by flanking zinc finger domains. These findings disfavor the utility of existing simple recognition codes and suggest that highly specific domains cannot be obtained from phage display alone in most cases, but only in combination with rational design. The molecular basis of zinc finger/DNA interaction is complex and its understanding is dependent on the analysis of a large number of proteins. This understanding should enable us to refine rapidly the specificity of other zinc finger domains, as well as polydactyl proteins constructed with these domains to recognize extended DNA sequences.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , Engenharia de Proteínas , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Simulação por Computador , Cristalografia por Raios X , DNA/química , Proteínas de Ligação a DNA/genética , Desenho de Fármacos , Histidina/genética , Histidina/metabolismo , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Biblioteca de Peptídeos , Maleabilidade , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
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 , EstereoisomerismoRESUMO
Phage display has become an important approach for the preparation of monoclonal antibodies from both immune and nonimmune sources. This approach allows for the rapid selection of monoclonal antibodies without the restraints of the conventional hybridoma approach. Although antibodies to a wide variety of antigens have been selected using phage display, some highly conserved mammalian antigens have proven to be less immunogenic in mammalian animals commonly used for immunization. In order to optimize methods for constructing chicken immunoglobulin phage display libraries in the pComb3 system, we have immunized chickens with the hapten fluorescein, and generated combinatorial antibody libraries from spleen and bone marrow RNA. Herein we present methods for the isolation of scFv, diabody and Fab fragment libraries from chickens. Chicken Fab fragment libraries are constructed using human constant regions, facilitating detection with readily available reagents as well as humanization. Analysis of the selected V-genes revealed that gene conversion events were more extensive in light-chain variable region genes as compared to heavy-chain variable region genes. In addition, we present a new variant of the pComb3 phage display vector system.
Assuntos
Anticorpos Monoclonais/biossíntese , Fragmentos de Imunoglobulinas/biossíntese , Biblioteca de Peptídeos , Animais , Sequência de Bases , Galinhas , Fluoresceína , Humanos , Imunização , Fragmentos Fab das Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/biossíntese , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Análise de Sequência de DNA/métodos , Homologia de Sequência de AminoácidosRESUMO
We describe the isolation of a CCR5-specific antibody, ST6, from an antibody phage display library generated from an immune rabbit. ST6 was previously shown to efficiently prevent the surface expression of CCR5 when expressed intracellularly (Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B. E., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 805-810). Because ST6 has therapeutic potential in human immunodeficiency virus, type 1 disease, its humanization was desired to minimize the potential for immunogenicity. ST6 was humanized using a phage display-based approach. Like the parental rabbit clone, the humanized version ST6/34 efficiently prevented the surface expression of CCR5. The conserved linear peptide epitope bound by these antibodies was mapped using phage display. Both ST6 as well as the humanized anti-CCR5 antibody ST6/34 were produced as complete IgG antibodies and shown to bind to cell surface CCR5.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Especificidade de Anticorpos , Engenharia de Proteínas , Receptores CCR5/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Anticorpos/uso terapêutico , Sequência de Bases , Antagonistas dos Receptores CCR5 , Quimiocinas/metabolismo , Mapeamento de Epitopos , Terapia Genética , HIV-1/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/uso terapêutico , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/uso terapêutico , Dados de Sequência Molecular , Biblioteca de Peptídeos , Coelhos , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores de HIV/antagonistas & inibidores , Receptores de HIV/genética , Receptores de HIV/imunologia , Receptores de HIV/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Alinhamento de SequênciaRESUMO
Ligand-dependent transcriptional regulators were generated by fusion of designed Cys(2)-His(2) zinc finger proteins and steroid hormone receptor ligand binding domains. To produce novel DNA binding domains, three-finger proteins binding specific 9-base pair sequences were constructed from modular building blocks. Fusion of these zinc finger proteins to a transcriptional activation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent transcriptional regulators. Together with optimized minimal promoters, these regulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expression systems are functionally independent, and each can be selectively switched on within the same cell. The potential use of zinc finger-steroid receptor fusion proteins for the regulation of natural promoters was also explored. A gene-specific six-finger protein binding an 18-base pair target sequence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecular dimerization and are functional as monomers. Thus, the ability to engineer DNA binding specificities of zinc finger proteins enables the construction of ligand-dependent transcriptional regulators with potential for the regulation of virtually any desired artificial or natural promoter. It is anticipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specific modulation of gene expression can be exploited.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Variação Genética , Humanos , Ligantes , Luciferases/biossíntese , Luciferases/genética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Ácido Nucleico , Receptores X de Retinoides , Especificidade por Substrato , TATA Box , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.
Assuntos
Anticorpos/genética , Anticorpos/uso terapêutico , Especificidade de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Afinidade de Anticorpos , Sequência de Bases , Biblioteca Gênica , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Coelhos , Especificidade da EspécieRESUMO
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.