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1.
Sci Rep ; 9(1): 18568, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31811202

RESUMO

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.


Assuntos
Proteínas do Capsídeo/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Neoplasias/terapia , Oligopeptídeos/metabolismo , Transdução Genética , Adenoviridae/genética , Adenoviridae/metabolismo , Motivos de Aminoácidos/genética , Proteínas do Capsídeo/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Receptores ErbB/metabolismo , Fibroblastos , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Dispositivos Lab-On-A-Chip , Neoplasias/genética , Esferoides Celulares , Internalização do Vírus
2.
J Mol Biol ; 312(5): 1059-71, 2001 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-11580250

RESUMO

Class I major histocompatibility complex (MHC) molecules, which display intracellularly processed peptides on the cell surface for scanning by T-cell receptors (TCRs), are extraordinarily polymorphic. MHC polymorphism is believed to result from natural selection, since individuals heterozygous at the corresponding loci can cope with a larger number of pathogens. Here, we present the crystal structures of the murine MHC molecule H-2D(b) in complex with the peptides gp276 and np396 from the lymphocytic choriomeningitis virus (LCMV), solved at 2.18 A and 2.20 A resolution, respectively. The most prominent feature of H-2D(b) is a hydrophobic ridge that cuts across its antigen-binding site, which is conserved in the L(d)-like family of class I MHC molecules. The comparison with previously solved crystal structures of peptide/H-2D(b) complexes shows that the hydrophobic ridge focuses the conformational variability of the bound peptides in a "hot-spot", which could allow optimal TCR interaction and discrimination. This finding suggests a functional reason for the conservation of this structural element.


Assuntos
Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos H-2/química , Antígenos H-2/imunologia , Vírus da Coriomeningite Linfocítica/química , Vírus da Coriomeningite Linfocítica/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Evolução Molecular , Antígeno de Histocompatibilidade H-2D , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia
3.
Trends Biochem Sci ; 26(10): 577-9, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11589999

RESUMO

Currently, the combination of library selection and directed evolution is the most powerful approach for finding proteins with novel folds or functions. In the past, most studies concentrated either on protein scaffolds with a given fold or on short peptides. With the recent development of potent in vitro selection and evolution techniques, the screening of much larger sequence space is possible, allowing for the de novo generation of proteins.


Assuntos
Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Evolução Molecular Direcionada , Técnicas In Vitro , Biblioteca de Peptídeos
4.
Curr Opin Biotechnol ; 12(4): 400-5, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11551470

RESUMO

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.


Assuntos
DNA Complementar/genética , Biblioteca Gênica , Proteínas/genética , RNA Mensageiro/genética , Ribossomos/genética , Sítios de Ligação/fisiologia , DNA Complementar/metabolismo , Evolução Molecular Direcionada/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade Enzimática/fisiologia , Mutagênese/genética , Biblioteca de Peptídeos , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
5.
J Mol Biol ; 310(2): 485-98, 2001 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-11428902

RESUMO

We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity. We have now investigated the molecular mechanism of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site. The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site. Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520. Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain. The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity.


Assuntos
Escherichia coli/enzimologia , Imunofilinas/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Tacrolimo/análogos & derivados , Sítios de Ligação , Ligação Competitiva , Catálise , Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Sequência Conservada , Dimerização , Proteínas de Escherichia coli , Haemophilus influenzae/enzimologia , Imunofilinas/antagonistas & inibidores , Imunofilinas/química , Isomerismo , Cinética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/química , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/química , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Renaturação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ribonuclease T1/química , Ribonuclease T1/metabolismo , Tacrolimo/metabolismo , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Termodinâmica
6.
J Biol Chem ; 276(17): 14385-92, 2001 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-11278961

RESUMO

Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185(HER-2) single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185(HER-2) and on SK-OV-3 cells. The (99m)technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.


Assuntos
Anticorpos/imunologia , Peptídeos/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Animais , Anticorpos/metabolismo , Cromatografia em Gel , Clonagem Molecular , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Feminino , Humanos , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Periplasma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Radioimunoensaio , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/metabolismo , Tecnécio/farmacocinética , Temperatura , Fatores de Tempo , Distribuição Tecidual , Células Tumorais Cultivadas
7.
J Mol Biol ; 305(5): 1111-29, 2001 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-11162118

RESUMO

Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.


Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Dobramento de Proteína , Anticorpos Monoclonais/imunologia , Humanos , Hidrogênio/metabolismo , Região Variável de Imunoglobulina/imunologia , Isomerismo , Cinética , Modelos Moleculares , Prolina/química , Prolina/metabolismo , Desnaturação Proteica , Renaturação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor ErbB-2/imunologia , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica
8.
J Immunol Methods ; 236(1-2): 147-65, 2000 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-10699587

RESUMO

The very low affinity of the T-cell receptor (TCR) for the peptide-major histocompatibility complex (pMHC) has made it very challenging to design assays for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and refolding methodologies for the TCR. We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs. It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientation to an immobilized anti-Cbeta antibody. Specificity can be stringently demonstrated by inhibition with monomeric pMHCs. The assay is very sensitive and specific, and requires only very small amounts of protein. It has allowed us to study the unstable recombinant TCR P14, which we expressed and refolded from Escherichia coli. The TCR P14 is directed against the most abundant epitope of LCMV. We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the interaction of the P14 TCR and of the gp33-pMHC as 6 microM. This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinity thus does not seem to be the cause of the modest protective power of these T-cells, compared to others elicited in the anti-LCMV response. This strategy of multimerizing one partner and immobilizing the other in both a native form and productive orientation should be generally useful for characterizing the weak interactions of cell-surface molecules.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Escherichia coli/genética , Expressão Gênica , Vírus da Coriomeningite Linfocítica/imunologia , Complexo Principal de Histocompatibilidade , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade
9.
Eur J Biochem ; 267(3): 627-33, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10651797

RESUMO

Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.


Assuntos
Amiloidose/diagnóstico por imagem , Diálise Renal , Uremia/diagnóstico por imagem , Microglobulina beta-2/biossíntese , Amiloidose/complicações , Sequência de Bases , Cromatografia de Afinidade , Primers do DNA/genética , Escherichia coli/genética , Humanos , Radioisótopos de Índio , Radioisótopos do Iodo , Masculino , Pessoa de Meia-Idade , Peso Molecular , Ácido Pentético/análogos & derivados , Controle de Qualidade , Cintilografia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Uremia/complicações , Microglobulina beta-2/genética
10.
Cancer Res ; 59(22): 5758-67, 1999 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-10582696

RESUMO

The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.


Assuntos
Anticorpos Antineoplásicos/química , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Temperatura Alta , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Receptor ErbB-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/isolamento & purificação , Anticorpos Antineoplásicos/metabolismo , Especificidade de Anticorpos , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Fragmentos de Imunoglobulinas/isolamento & purificação , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/metabolismo , Marcação por Isótopo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Químicos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptor ErbB-2/metabolismo , Alinhamento de Sequência , Tecnécio , Células Tumorais Cultivadas
11.
FEBS Lett ; 459(2): 166-72, 1999 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-10518012

RESUMO

We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.


Assuntos
beta-Lactamases/química , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Conformação Proteica , Desnaturação Proteica , Homologia de Sequência de Aminoácidos
12.
Nat Biotechnol ; 17(9): 897-901, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10471933

RESUMO

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.


Assuntos
Histidina/química , Marcação por Isótopo/métodos , Compostos de Organotecnécio/síntese química , Compostos Radiofarmacêuticos/síntese química , Proteínas Recombinantes/química , Tecnécio , Aldeídos , Animais , Anticorpos/genética , Anticorpos/imunologia , Cromatografia de Afinidade , Humanos , Fragmentos de Imunoglobulinas/química , Cetonas , Camundongos , Camundongos Nus , Mucina-1/imunologia , Compostos de Organotecnécio/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes/farmacocinética , Distribuição Tecidual
14.
Curr Opin Struct Biol ; 9(4): 514-20, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10449374

RESUMO

Phage display of a wide range of polypeptides has been increasingly used to identify novel molecules with useful binding properties for research, medical and industrial applications. Recent developments include methods for the selection of stabilized variants of a protein, the selection of regulatable enzymes and promising strategies for the selection and evolution of protein catalysts.


Assuntos
Bacteriófagos/química , Catálise , Clonagem Molecular/métodos , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas/métodos , Dobramento de Proteína , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Seleção Genética
15.
Nat Biotechnol ; 17(7): 683-90, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10404162

RESUMO

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.


Assuntos
Escherichia coli/enzimologia , Zíper de Leucina , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Dobramento de Proteína , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Dimerização , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Camundongos , Mutação , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética
16.
J Mol Biol ; 285(4): 1831-43, 1999 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-9917415

RESUMO

In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions. The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10(-4) M. Using T-cell receptor constructs comprising all extracellular domains (ValphaCalpha and VbetaCbeta), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Calpha-Cbeta disulfide bridge bond was deleted, even though the interaction between the complete alpha and beta-chain was determined to be very weak and fully reversible (KD approximately 10(-7) to 10(-6) M). Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation. Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated. This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation. Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Animais , Dimerização , Escherichia coli/genética , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D , Técnicas In Vitro , Corpos de Inclusão/química , Ligantes , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Ressonância de Plasmônio de Superfície
17.
J Immunol Methods ; 231(1-2): 93-104, 1999 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-10648930

RESUMO

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology.


Assuntos
Bacteriófago M13/genética , Proteínas de Ligação a DNA/genética , Biblioteca de Peptídeos , Proteínas Virais de Fusão/genética , Bacteriófago M13/fisiologia , Proteínas do Capsídeo , Humanos , Recombinação Genética
18.
J Mol Biol ; 283(1): 95-110, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9761676

RESUMO

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.


Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Substituição de Aminoácidos , Dissulfetos/química , Ligação de Hidrogênio , Fragmentos de Imunoglobulinas/química , Indanos/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica , Serina/química , Ureia
19.
Nat Biotechnol ; 16(10): 955-60, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9788353

RESUMO

We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.


Assuntos
Inovirus/genética , Ribonuclease T1/genética , Sequência de Bases , Proteínas do Capsídeo , Primers do DNA , Proteínas de Ligação a DNA/genética , Hidrólise , Inovirus/patogenicidade , Mutagênese Sítio-Dirigida , Ribonuclease T1/metabolismo , Termodinâmica , Proteínas Virais de Fusão/genética
20.
Biochemistry ; 37(38): 13120-7, 1998 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-9748318

RESUMO

A set of six mutants of the levan binding single-chain Fv (scFv) fragment A48 (ABPC48), which have the identical light chain but differ gradually in the stability of the heavy chain, was generated. This was achieved by introducing one or both of the stabilizing mutations H-K66R and H-N52S into the VH domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in VH, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in VH, which had been selected in the context of a disulfide-free derivative of this scFv fragment [Proba, K., et al. (1998) J. Mol. Biol. 275, 245-253], were found to be additive and transferable to the cysteine-restored variant of the A48 scFv, thereby generating extremely stable VH domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated VL domain and two of the different isolated VH domains. In the scFv fragment, the VL domain was found to be stabilized by a more stable VH domain, and, conversely, the VH domain was stabilized by a more stable VL domain. A folding intermediate with nativelike VH and denatured VL was found at equilibrium, if VH was significantly more stable than VL. In all other cases, a cooperative unfolding of the scFv was observed. We explain this observation with different contributions of intrinsic domain stability and extrinsic stabilization provided by the partner domain in the single-chain antibodies.


Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Engenharia de Proteínas , Hidrólise , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Termolisina/metabolismo
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