RESUMEN
We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.
Asunto(s)
Mutación de Línea Germinal , Región Variable de Inmunoglobulina/genética , Recombinación Genética , Humanos , Fragmentos de Inmunoglobulinas/genética , Proteínas Recombinantes/genéticaRESUMEN
Non-glycine residues in proteins are rarely observed to have "left-handed helical" conformations. For glycine, however, this conformation is common. To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine. The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity. The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme. The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/mol). These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar. This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol. Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure. In the case of the Asn55 to Gly replacement, however, the (phi, psi) values of residue 55 change by about 20 degrees. This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues. This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (phi = 90 degrees, psi = 0 degrees), whereas that for non-glycine residues is close to (phi = 60 degrees, psi = 30 degrees). This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (phi, psi) energy contour maps.
Asunto(s)
Muramidasa , Fagos T/enzimología , Proteínas Virales , Secuencia de Aminoácidos , Asparagina , Cristalografía , Estabilidad de Enzimas , Glicina , Lisina , Datos de Secuencia Molecular , Muramidasa/metabolismo , Mutación , Conformación Proteica , Termodinámica , Proteínas Virales/metabolismoRESUMEN
Proteins and peptides can be displayed on bacterial and bacteriophage surfaces as fusions to bacterial integral membrane proteins or phage coat proteins. We now report on the expression of peptide antigens on the surface of F pili, elaborated by F+ strains of Escherichia coli. The peptides were expressed as fusions to F pilin, the building block of the F pilus that is encoded by the traA gene on the F plasmid. Filamentous bacteriophage infection of E. coli is normally mediated by phage binding to pilin at the F pili tip. Expression of 13 to 15 amino acid long peptides on the F pilus completely blocked infection by derivatives of wild-type infectious M13 phage. However, when a phage displaying a specific recombinant antibody fragment was allowed to interact with F pili displaying an antigenic peptide a bacterial infection could be demonstrated. This infection, mediated by the antibody-antigen interaction, resulted in bacterial cells containing plasmids encoding both the protein and the ligand. In a model library, where a scFv antibody against the human cytomegalovirus AD-2 epitope was selected we achieved an enrichment of 2500 of phage carrying the specific antibody, indicating an efficient selective infection.
Asunto(s)
Antígenos Virales/genética , Proteínas de la Membrana Bacteriana Externa/genética , Bacteriófago M13/fisiología , Proteínas de Escherichia coli , Escherichia coli/virología , Pili Sexual/genética , Anticuerpos Antivirales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Bacteriófago M13/inmunología , Bacteriófago M13/ultraestructura , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Escherichia coli/genética , Escherichia coli/ultraestructura , Proteínas Fimbrias , Proteína gp120 de Envoltorio del VIH/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Fragmentos de Péptidos/genética , Biblioteca de Péptidos , Pili Sexual/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Muramidasa/química , Fagos T/enzimología , Arginina/química , Electroquímica , Estabilidad de Enzimas , Enlace de Hidrógeno , Lisina/química , Modelos Moleculares , Muramidasa/genética , Muramidasa/metabolismo , Mutagénesis Sitio-Dirigida , Fagos T/genética , Temperatura , Difracción de Rayos XRESUMEN
A completely synthetic gene library encoding the variable light (VL) immunoglobulin domains has been constructed in vitro. The library was constructed by assembling a set of six oligodeoxyribonucleotides (oligos) using the polymerase chain reaction (PCR). Three out of the six overlapping oligonucleotides were synthesized with randomized complementarity determining regions (CDR) with the codon pattern, (NNS)n, where N is any of the four nucleotides (nt) and n is the number of codons with variation in the CDR. The framework regions, taken from the D1.3 anti-lysozyme antibody (Ab), were kept intact. Overlapping regions of approx. 20 nt, together with two additional flanking primers carrying the desired restriction sites, allowed the construction of a library in one single PCR reaction. The VL library was cloned into the phage display vector pEXmide3, and ten randomly picked clones were sequenced. These sequences exhibited complete diversity in all the three CDR and the codons for five canonical amino acid (aa) residues were kept intact and identified. Seven clones contained the full-length gene for the VL domain while deletions were observed in three clones. The restricted use of nt at the third position successfully avoided the stop codons TGA and TAA, whereas the stop codon TAG is read as Gln in an amber suppressor strain. We call this synthetic Ab diversity Domain Library, and it represents an example of synthetic libraries with extensive, multiple randomized sequences. The use of Domain Libraries opens up the possibility for design in Ab engineering, e.g., additional CDR regions can be added or their length varied.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Diversidad de Anticuerpos/genética , Biblioteca de Genes , Genes de Inmunoglobulinas , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Anticuerpos/química , Anticuerpos/inmunología , Secuencia de Bases , Clonación Molecular , Codón/genética , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Muramidasa/inmunología , Reacción en Cadena de la Polimerasa , Distribución AleatoriaRESUMEN
A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.
Asunto(s)
Secuencia de Bases , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Modelos Genéticos , Alineación de Secuencia , Secuencia de Aminoácidos , Clonación Molecular , Cartilla de ADN , Escherichia coli , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de AminoácidoRESUMEN
Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable region was shown to belong to the V kappa V family and contained a previously unreported Ile72. Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain. When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment. This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA. The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment. Together, this suggested that the translation process was involved in the restricted production of the L-chain. Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phage-displayed Ab libraries.
Asunto(s)
Escherichia coli/genética , Región Variable de Inmunoglobulina/genética , Mutación Puntual , Proteínas Recombinantes/genética , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Escherichia coli/inmunología , Vectores Genéticos , Fragmentos Fab de Inmunoglobulinas/genética , Isoleucina/genética , Datos de Secuencia Molecular , Operón , ARN Mensajero/genéticaRESUMEN
In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.
Asunto(s)
Bacteriófagos/metabolismo , Sitios de Unión de Anticuerpos/inmunología , Técnicas Biosensibles/clasificación , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Secuencia de Bases/genética , Células Clonales , Biblioteca de Genes , Fragmentos Fab de Inmunoglobulinas/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Cinética , Muramidasa/inmunología , Unión Proteica/inmunologíaRESUMEN
In this work, we have investigated parameters important for the assembly of synthetic genes encoding antibody fragments. These genes are constructed from a set of overlapping single-stranded oligonucleotides (primers), which are assembled into a gene sequence in a one-step process using PCR. Using the Oligo program, we made a detailed analysis of wanted and unwanted interactions between these primers; both the stability of hairpin structures of homodimers and of heterodimers were examined. The Oligo program could be used to identify unwanted interactions of high stability, and the present study suggests that if the stabilities of the unwanted interactions are kept to 25%-50% of the designed interactions, a successful assembly of synthetic genes can occur.
Asunto(s)
Especificidad de Anticuerpos , Expresión Génica , Fragmentos de Inmunoglobulinas/genética , Secuencia de Bases , ADN/biosíntesis , Cartilla de ADN , Estabilidad de Medicamentos , Biblioteca de Genes , Humanos , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Datos de Secuencia Molecular , Oligonucleótidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter. For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector. The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding. Co-expression of chaperonin-encoding plasmid pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein twofold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/genética , Expresión Génica , Proteínas de Choque Térmico/fisiología , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas , Proteínas Bacterianas/genética , Western Blotting , Antígeno Carcinoembrionario/inmunología , Chaperonina 10 , Chaperonina 60 , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/genética , Técnicas para Inmunoenzimas , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/metabolismo , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la EspecieRESUMEN
The n-CoDeR recombinant antibody gene libraries are built on a single master framework, into which diverse in vivo-formed complementarity determining regions (CDRs) are allowed to recombine. These CDRs are sampled from in vivo-processed and proof-read gene sequences, thus ensuring an optimal level of correctly folded and functional molecules. By the modularized assembly process, up to six CDRs can be varied at the same time, providing a possibility for the creation of a hitherto undescribed genetic and functional variation. The n-CoDeR antibody gene libraries can be used to select highly specific, human antibody fragments with specificities to virtually any antigen, including carbohydrates and human self-proteins and with affinities down into the subnanomolar range. Furthermore, combining CDRs sampled from in vivo-processed sequences into a single framework result in molecules exhibiting a lower immunogenicity compared to normal human immunoglobulins, as determined by computer analyses. The distinguished features of the n-CoDeR libraries in the therapeutic and diagnostic areas are discussed.
Asunto(s)
Anticuerpos/genética , Biblioteca de Genes , Animales , Anticuerpos/uso terapéutico , Humanos , Fragmentos de Inmunoglobulinas/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Technologies to develop and evolve the function of proteins and, in particular, antibodies have developed rapidly since the introduction of phage display. Importantly, it has become possible to identify molecules with binding properties that cannot be found by other means. A range of different approaches to create general libraries that are useful for the selection of such molecules specific for essentially any kind of target have emerged. We herein review some of the most prominent approaches in the field and in particular discuss specific features related to the development of antibody libraries based on single antibody framework scaffolds. This approach not only permits identification of a range of specific binders, but also facilitates further evolution of initially derived molecules into molecules with optimised functions.
Asunto(s)
Anticuerpos/química , Técnicas Químicas Combinatorias , Proteínas/química , Afinidad de Anticuerpos , Regiones Determinantes de ComplementariedadRESUMEN
We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3. Titers of the packed phagemid increased almost 200-fold from approximately 4 x 10(11) cfu/ml, without coexpression of the GroE proteins, to approximately 7 x 10(13) cfu/ml with their coexpression. Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments. While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments. pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains. The vector allows cloning of genes for the variable domains linking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains. A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain.
Asunto(s)
Proteínas Bacterianas/farmacología , Bacteriófagos/genética , Vectores Genéticos , Proteínas de Choque Térmico/farmacología , Fragmentos Fab de Inmunoglobulinas/genética , Proteínas Bacterianas/genética , Bacteriófagos/inmunología , Secuencia de Bases , Chaperonina 10 , Chaperonina 60 , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Expresión Génica , Proteínas de Choque Térmico/genética , Datos de Secuencia Molecular , Plásmidos , Mapeo RestrictivoRESUMEN
Magnetic marker monitoring (MMM) is a new technique for visualizing transit and disintegration of solid oral dosage forms through the gastrointestinal (GI) tract. The aim of this work was to develop a modeling approach for gaining information from MMM studies using data from a food interaction study with felodipine extended-release (ER) formulation. The interrelationship between tablet location in the GI tract, in vivo drug release, and felodipine disposition was modeled. A Markov model was developed to describe the tablet's movement through the GI tract. Tablet location within the GI tract significantly affected drug release and absorption through the gut wall. Food intake decreased the probability of tablet transition from the stomach, decreased the rate with which released felodipine left the stomach, and increased the fraction absorbed across the gut wall. In conclusion, the combined information of tablet location in the GI tract, in vivo drug release, and plasma concentration can be utilized in a mechanistically informative way with integrated modeling of data from MMM studies.
Asunto(s)
Biomarcadores/metabolismo , Tránsito Gastrointestinal/efectos de los fármacos , Tránsito Gastrointestinal/fisiología , Magnetismo , Modelos Biológicos , Preparaciones Farmacéuticas/metabolismo , Administración Oral , Biomarcadores/análisis , Estudios Cruzados , Humanos , Cadenas de Markov , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/análisis , ComprimidosRESUMEN
The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.
Asunto(s)
Asparagina/química , Ácido Aspártico/química , Ribulosa-Bifosfato Carboxilasa/química , Sitios de Unión , Cristalización , Sustancias Macromoleculares , Magnesio/metabolismo , Estructura Molecular , Mutagénesis Insercional , Conformación Proteica , Rhodospirillum rubrum/enzimología , Ribulosa-Bifosfato Carboxilasa/genética , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
A novel technology in the area of antibody engineering has been developed which allows for the creation of new types of antibody molecules. It is called complementarity-determining region (CDR) implantation and permits the random combination of CDR sequences formed in vivo into a single master framework. Thus, totally new gene combinations can be produced and used in selection processes. The result is a genetic variability which is extremely large, even exceeding the natural variability found in the immune system. In this commentary, CDR implantation is presented and the technology is discussed.
Asunto(s)
Anticuerpos/genética , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas/métodos , Animales , Anticuerpos/inmunología , Variación Genética , Humanos , Recombinación GenéticaRESUMEN
Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.
Asunto(s)
Coagulación Sanguínea/genética , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Deleción Cromosómica , Ensayo de Inmunoadsorción Enzimática , Femenino , Ligamiento Genético/genética , Variación Genética/genética , Humanos , Masculino , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Suecia , Enfermedades de von Willebrand/sangreRESUMEN
The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the mutant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (epsilon max = 11400 M-1 cm-1). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F mutant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme [Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619]. This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the mutant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F mutant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the mutant of 0.74 s-1 versus 20 s-1 for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave Km values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The Km for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the Km for oxygen is much lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s-1. The kinetic properties of Y129F glycolate oxidase provide evidence that the main function of the hydroxyl group of Tyr129 is the stabilization of the transition state.
Asunto(s)
Oxidorreductasas de Alcohol/química , Tirosina/química , Oxidorreductasas de Alcohol/metabolismo , Sitios de Unión , Cristalización , Glicolatos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Plantas/enzimología , Relación Estructura-ActividadRESUMEN
Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.