RESUMO
Correction for 'Combination of inverse electron-demand Diels-Alder reaction with highly efficient oxime ligation expands the toolbox of site-selective peptide conjugations' by S. Hörner, et al., Chem. Commun., 2015, DOI: 10.1039/c5cc03434e.
RESUMO
A modular approach combining inverse electron-demand Diels-Alder coupling (DARinv) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.
Assuntos
Elétrons , Oximas/química , Peptídeos/química , Cistina/química , Dissulfetos/química , Estrutura Molecular , Nanopartículas/química , Compostos de Organossilício/químicaRESUMO
It was the aim of this study to evaluate the stability of two novel cystine-knot microproteins (CKM) SE-ET-TP-020 and SE-MC-TR-020 with potential clinical relevance towards luminally secreted proteases of the gastrointestinal tract in order to gain information about their potential for oral administration. Therefore, the stability of the two CKM and the model-drug insulin towards collected porcine gastric and small intestinal juice as well as towards isolated proteolytic enzymes was evaluated under physiological conditions. No intact SE-ET-EP-020 was detected after few seconds of incubation with porcine small intestinal juice. SE-ET-TP-020 was also degraded in porcine gastric juice. Furthermore, SE-ET-TP-020 was extensively degraded by isolated chymotrypsin, trypsin and pepsin. Moreover, it was degraded by elastase. SE-MC-TR-020 was degraded entirely within approximately 2 h when incubated in porcine small intestinal juice, whereas no degradation was observed within a 3 h incubation period with porcine gastric juice. In presence of the isolated proteolytic enzymes, SE-MC-TR-020 was only slightly degraded by trypsin and pepsin, whereas elastase caused no degradation to SE-MC-TR-020 at all. Chymotrypsin was the protease that caused most degradation to SE-MC-TR-020. The model drug insulin was degraded extensively by chymotrypsin, elastase, pepsin and trypsin as well as by porcine gastric and porcine small intestinal juice. In conclusion, a precise characterisation of SE-ET-TP-020 and SE-MC-TR-020 degrading luminally secreted GI enzymes has been made, which is an important and substantial prerequisite for the further optimisation of these CKM.
Assuntos
Suco Gástrico/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Secreções Intestinais/química , Peptídeo Hidrolases/química , Animais , Insulina/química , Suínos , Fatores de TempoRESUMO
Cystine-knot microproteins exhibit several properties that make them highly interesting as scaffolds for oral peptide drug delivery. It was therefore the aim of the study to evaluate the novel clinically relevant cystine-knot microprotein McoEeTI regarding its potential for oral delivery. Additionally, based on the gained results, important features of McoEeTI were improved. Enzymatic degradation was caused by chymotrypsin, trypsin and porcine small intestinal juice whereas McoEeTI was stable towards elastase, membrane bound proteases, pepsin and porcine gastric juice. Only minor McoEeTI degradation was observed during a 24h incubation period in rat plasma. In the presence of various physiological ions about 50% of McoEeTI formed di- and/or trimers. P(app) value of McoEeTI was determined to be (7.4+/-0.4)x10(-6)cm/s. Sodium caprate and polycarbophil-cysteine (PCP-Cys) had no beneficial effect on McoEeTI permeation, whereas the utilization of a chitosan-thiobutylamidine (Chito-TBA) system improved McoEeTI permeation 3-fold. Enzymatic stability could be strongly improved by the utilization of Bowman-Birk-Inhibitor (BBI) as well as PCP-Cys. In conclusion, this study indicates that McoEeTI represents a promising candidate as a novel scaffold for oral peptide drug delivery.
Assuntos
Ciclotídeos/química , Motivos Nó de Cisteína , Portadores de Fármacos , Proteínas de Plantas/química , Proteínas Recombinantes de Fusão/química , Resinas Acrílicas/química , Administração Oral , Sequência de Aminoácidos , Animais , Permeabilidade da Membrana Celular , Quitina/análogos & derivados , Quitina/química , Quimotripsina/química , Quimotripsina/metabolismo , Ciclotídeos/genética , Cisteína/química , Cistina/química , Cistina/genética , Cultura em Câmaras de Difusão , Estabilidade de Medicamentos , Absorção Intestinal , Dados de Sequência Molecular , Proteínas de Plantas/genética , Conformação Proteica , Engenharia de Proteínas , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Serina/genética , Suínos , Fatores de Tempo , Tripsina/química , Tripsina/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/químicaRESUMO
We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.
Assuntos
Anticorpos/isolamento & purificação , Mapeamento de Epitopos/métodos , Escherichia coli/genética , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/imunologia , Cricetinae , DNA Recombinante/genética , Escherichia coli/imunologia , Citometria de Fluxo , Vetores Genéticos , Dados de Sequência Molecular , Transfecção , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.
Assuntos
Adesinas Bacterianas/biossíntese , Proteínas de Transporte/biossíntese , Escherichia coli O157 , Proteínas de Escherichia coli , Escherichia coli , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Aderência Bacteriana , Proteínas de Transporte/genética , Epitopos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Vírus Sendai/imunologiaRESUMO
The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA', which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.
Assuntos
Cistina/química , Escherichia coli/química , Peptídeos/química , Inibidores de Proteases/química , Verduras/química , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/química , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22-25 comprising a beta-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22-25 tetrapeptide sequences and presented these proteins on the outer membrane of Escherichia coli cells via fusion to the Iga(beta) autotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1-5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in beta-turns. The folding pathway of two selected variants, EETI-beta(NEDE) and EETI-beta(TNNK), was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.
Assuntos
Proteínas de Plantas , Plantas/genética , Inibidores da Tripsina/genética , Escherichia coli , Biblioteca Gênica , Plantas/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismoRESUMO
ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (OR1 PR-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli. However, in E. coli, ToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of lambda-ToxR chimeras. When the OR1 PR-lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E. coli suggested that lambda-ToxR dimers could act co-operatively. Using a redesigned reporter system containing multiple lambda operator sites (OR1 OR2 OR3 PR-lacZY), we found that lambda-ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co-operative interactions. However, we falled to correlate a monomer-dimer transition of the lambda-ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. cholerae, deletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild-type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Conformação Proteica , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Western Blotting , Proteínas de Ligação a DNA/genética , Dimerização , Escherichia coli , Genes Reporter , Concentração de Íons de Hidrogênio , Óperon Lac , Proteínas de Membrana/genética , Concentração Osmolar , Periplasma , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Fatores de Transcrição/genética , Vibrio cholerae , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
Specific interactions between membrane spanning polypeptide segments are important for folding and oligomerisation of integral membrane proteins. Previously the dimerisation of glycophorin A has been shown to depend on interactions between its transmembrane segment by studying chimeric proteins in detergent solution. Here, we examined dimerisation of the glycophorin A transmembrane segment in a natural membrane employing the ToxR transcription activator from Vibrio cholerae. The ToxR protein is integral to the bacterial inner membrane and its activity requires a dimeric state. Therefore, the ToxR protein is suited to monitor quantitative homophilic interactions. We replaced the ToxR transmembrane segment with parts of the glycophorin A transmembrane segment containing the amino acid motif LIxxGVxxGVxxT previously shown to be sufficient for dimerisation in detergent solution. Expression of these chimeric proteins in an indicator strain resulted in strong transcription activation. This is indicative of efficient dimerisation mediated by the glycophorin transmembrane segment inserted into the inner membrane. Analysis of individual point mutants revealed that at least four residues out of this motif are critical for dimer formation in membranes. However, dimerisation of the glycophorin A transmembrane segment appears to be less sensitive to mutations when localised within a natural lipid bilayer compared to measurements in detergent solution. This may be related to a slightly altered structure of the dimer and/or to a higher local concentration and preorientation of the interacting molecules in a membrane. This makes the ToxR system well suited for probing low-affinity interactions between the transmembrane segments of other proteins.
Assuntos
Proteínas de Bactérias , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoforinas/química , Glicoforinas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoforinas/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
DegP and DegQ are homologous endoproteases found in the periplasmic compartment of Escherichia coli. The studies presented here suggest that DegP and DegQ have very similar substrate specificities and cleave substrates which are transiently or globally denatured. Model substrates were cleaved at discrete Val/Xaa or Ile/Xaa sites, suggesting that aliphatic, beta-branched residues, which are typically buried in the hydrophobic core of most proteins, are important determinants of cleavage specificity. Indeed, the peptide bonds cleaved in the model substrates are generally inaccessible in the native three-dimensional structures. In addition, a chimeric fusion protein, which is a DegP substrate in vivo, is degraded in vitro only after reduction of its intramolecular disulfide bonds. Taken together, these findings suggest that DegP and DegQ may degrade transiently denatured proteins, unfolded proteins which accumulate in the periplasm following heat shock or other stress conditions, and/or newly secreted proteins prior to folding and disulfide bond formation. Cross-linking studies indicate that both DegP and DegQ form dodecamers in solution and thus are similar to many other intracellular proteases which form large oligomeric complexes.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico , Proteínas Periplásmicas , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas , Dissulfetos/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND: Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues. RESULTS: Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed. CONCLUSIONS: A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.
Assuntos
Região Variável de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/química , Dobramento de Proteína , Dissulfetos , HumanosRESUMO
The Vibrio cholerae protein ToxR is an integral membrane protein that acts as a transcription activator in response to environmental signals; it controls expression of toxin genes ctxA and ctxB, along with a variety of other genes related to pathogenicity. Here it is shown that: (i) ToxR has a modular architecture and that activation of transcription starting at the ctx promoter depends strictly on dimerization of the periplasmic ToxR domain; (ii) the transmembrane (TM) region of ToxR is sufficient as a topogenic signal but not for stable membrane anchoring of the protein; (iii) the TM region has no special function in signal transduction and (iv) a proline residue located within the TM region minimizes background transcription activation, most plausibly by reducing TM-TM interaction. Possible applications of ToxR as a technical tool for analysing protein-protein interactions between pairs of arbitrary TM domains are discussed.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli , Proteínas de Membrana/química , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de Ligação , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Fatores de Transcrição/química , Ativação Transcricional/fisiologia , Vibrio cholerae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Toxina da Cólera/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Genes Bacterianos/genética , Proteínas Ligantes de Maltose , Proteínas de Membrana/genética , Dados de Sequência Molecular , Prolina/fisiologia , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição/genéticaRESUMO
A model repertoire of variants of immunoglobulin kappa variable domain REIv with different folding stabilities was generated by oligonucleotide-directed randomization of position 29, a key conserved residue of hypervariable loop 1. Fused to ToxR', the membrane-anchored cytoplasmic domain of the Vibrio cholerae ToxR transcription activator, different members of the library induce different levels of transcription from the ctx promoter in Escherichia coli. Differences in transcription activation correlate positively with folding stabilities of the corresponding REIv domains. Since conformationally stabilized REIv derivatives elicit a dark red colony phenotype on EMB-lactose indicator plates, this procedure constitutes a genetic screen for immunoglobulin folding stability.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Biblioteca Gênica , Cadeias kappa de Imunoglobulina/genética , Dobramento de Proteína , Transdução de Sinais , Fatores de Transcrição/genética , Vibrio cholerae/química , Sequência de Aminoácidos , Sequência de Bases , Toxina da Cólera/genética , Escherichia coli/genética , Genes Bacterianos/genética , Genes de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Ativação Transcricional , beta-Galactosidase/genética , beta-LactamasesRESUMO
Two amino acid exchanges (Y32H and C23V) were introduced sequentially into the immunoglobulin REIV, a human kappa variable domain. The first exchange stabilizes the folded state of the domain by 4.6 kJ/mol (1.1 kcal/mol), the second abolishes the central disulfide bridge and destabilizes the folded domain by 17.5 kJ/mol (4.2 kcal/mol). Introduction of the stabilizing exchange first is a necessary pre-requisite to the removal of the central disulfide bridge without collapse of the fold. The double mutant REIV-C23V/Y32H can be accumulated in the cytoplasmatic compartment of the E. coli cell, a finding that opens new possibilities in antibody engineering.
Assuntos
Região Variável de Imunoglobulina/química , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Dados de Sequência Molecular , Mutação/genética , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Espectrometria de FluorescênciaRESUMO
Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops. Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV). The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering. Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV. By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E. coli can be derived from REIV homodimer formation constant. The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E. coli chromosome. There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter. Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E. coli for immunoglobulin variants with altered association constants.
Assuntos
Proteínas de Bactérias , Proteína de Bence Jones/química , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Bases , Proteína de Bence Jones/genética , Cromatografia em Gel , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Polímeros , Proteínas Recombinantes de Fusão/química , Fatores de Transcrição/genética , Vibrio cholerae/genéticaRESUMO
A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.
Assuntos
Proteína de Bence Jones/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Região Variável de Imunoglobulina/genética , Mutagênese , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Plasmídeos , beta-Lactamases/genéticaRESUMO
In Escherichia coli K-12, the Dcm methyltransferase catalyses methylation of the inner cytosine residue in the sequence CCA/TGG. Hydrolytic deamination of 5-methylcytosine bases in DNA leads to thymine residues, and hence to T/G mismatches, pre-mutagenic DNA lesions consisting of two natural DNA constituents and thus devoid of an obvious marker of the damaged DNA strand. These mismatches are corrected by the VSP repair pathway, which is characterized by very short patches of DNA repair synthesis. It depends on genes vsr and polA and is strongly stimulated by mutL and mutS. The vsr gene product (Vsr; Mr 18,000) was purified and characterized as a DNA mismatch endonuclease, a unique and hitherto unknown type of enzyme. Vsr endonuclease nicks double-stranded DNA within the sequence CTA/TGN or NTA/TGG next to the underlined thymidine residue, which is mismatched to 2'-deoxyguanosine. The incision is mismatch-dependent and strand-specific. These results illustrate how Vsr endonuclease initiates VSP mismatch repair.
Assuntos
Reparo do DNA , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , 5-Metilcitosina , Composição de Bases , Sequência de Bases , Citosina/análogos & derivados , Citosina/metabolismo , Endodesoxirribonucleases/genética , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Timina/metabolismo , beta-Lactamases/genéticaRESUMO
Own investigations on in-vivo tumor metabolism of the malignant human colon tumor showed a significant uptake of branched chain amino acids by the tumor itself. To study the quantitative tumor protein metabolism ("compartment "tumor") the(13)C-leucine-tracer-technique was modified.Beside the common(13)C-leucine-breath-test we measured also the AV-differences of(13)C-leucine,(13)C-ketoisocaproate and(13)CO2. The "Tumorblood flow" was measured by "venous-outflow-technique" as well as the tumor mass.In this way it is possible to get quantitative results in substrate exchange of branched chain amino acids in malignant human colon tumors.