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1.
Bioorg Khim ; 29(5): 457-60, 2003.
Artigo em Russo | MEDLINE | ID: mdl-14601399

RESUMO

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217-221 of the enzyme are involved in formation of the binding pockets S3-S6. This indicates that the autolysis of the peptide bond Met218-Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in considerable decrease in the enzymatic activity.


Assuntos
Endopeptidases/química , Mutação , Potyvirus/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Endopeptidases/genética , Endopeptidases/metabolismo , Conformação Proteica
2.
Structure ; 9(12): 1225-36, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11738048

RESUMO

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.


Assuntos
Endorribonucleases/química , Proteínas de Escherichia coli , RNA de Cadeia Dupla/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Ligantes , Manganês/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribonuclease III , Homologia de Sequência de Aminoácidos
3.
J Mol Biol ; 312(4): 807-21, 2001 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-11575934

RESUMO

Many Gram-negative bacterial pathogens employ a contact-dependent (type III) secretion system to deliver effector proteins into the cytosol of animal or plant cells. Collectively, these effectors enable the bacteria to evade the immune response of the infected organism by modulating host-cell functions. YopM, a member of the leucine-rich repeat protein superfamily, is an effector produced by the bubonic plague bacterium, Yersinia pestis, that is essential for virulence. Here, we report crystal structures of YopM at 2.4 and 2.1 A resolution. Among all leucine-rich repeat family members whose atomic coordinates have been reported, the repeating unit of YopM has the least canonical secondary structure. In both crystals, four YopM monomers form a hollow cylinder with an inner diameter of 35 A. The domain that targets YopM for translocation into eukaryotic cells adopts a well-ordered, alpha-helical conformation that packs tightly against the proximal leucine-rich repeat module. A similar alpha-helical domain can be identified in virulence-associated leucine-rich repeat proteins produced by Salmonella typhimurium and Shigella flexneri, and in the conceptual translation products of several open reading frames in Y. pestis.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Leucina/metabolismo , Sequências Repetitivas de Aminoácidos , Yersinia pestis/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Cálcio/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Transporte Proteico , Reprodutibilidade dos Testes , Salmonella typhimurium/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Shigella flexneri/química , Água/química , Água/metabolismo , Yersinia enterocolitica/química
4.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 6): 793-9, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11375498

RESUMO

Yersinia pestis, the causative agent of bubonic plague, injects effector proteins into the cytosol of mammalian cells that enable the bacterium to evade the immune response of the infected organism by interfering with eukaryotic signal transduction pathways. YopH is a modular effector composed of a C-terminal protein tyrosine phosphatase (PTPase) domain and a multifunctional N-terminal domain that not only orchestrates the secretion and translocation of YopH into eukaryotic cells but also binds tyrosine-phosphorylated target proteins to mediate substrate recognition. The crystal structure of the N-terminal domain of YopH (YopH(N); residues 1-130) has been determined at 2.0 A resolution. The amino-acid sequences that target YopH for secretion from the bacterium and translocation into eukaryotic cells form integral parts of this compactly folded domain. The structure of YopH(N) bears no resemblance to eukaryotic phosphotyrosine-binding domains, nor is it reminiscent of any known fold. Residues that have been implicated in phosphotyrosine-dependent protein binding are clustered together on one face of YopH(N), but the structure does not suggest a mechanism for protein-phosphotyrosine recognition.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas Tirosina Fosfatases/química , Yersinia pestis/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição/química
5.
Protein Sci ; 10(3): 622-30, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11344330

RESUMO

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Substituição de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Transporte de Monossacarídeos , Mutagênese Sítio-Dirigida/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Substituição de Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Proteínas Ligantes de Maltose , Chaperonas Moleculares , Dobramento de Proteína , Solubilidade , Propriedades de Superfície
6.
J Mol Biol ; 305(4): 891-904, 2001 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-11162100

RESUMO

A maltodextrin-binding protein from Pyrococcus furiosus (PfuMBP) has been overproduced in Escherichia coli, purified, and crystallized. The crystal structure of the protein bound to an oligosaccharide ligand was determined to 1.85 A resolution. The fold of PfuMBP is very similar to that of the orthologous MBP from E. coli (EcoMBP), despite the moderate level of sequence identity between the two proteins (27 % identity, 46 % similarity). PfuMBP is extremely resistant to heat and chemical denaturation, which may be attributed to a number of factors, such as a tightly packed hydrophobic core, clusters of isoleucine residues, salt-bridges, and the presence of proline residues in key positions. Surprisingly, an attempt to crystallize the complex of PfuMBP with maltose resulted in a structure that contained maltotriose in the ligand-binding site. The structure of the complex suggests that there is a considerable energy gain upon binding of maltotriose in comparison to maltose. Moreover, isothermal titration calorimetry experiments demonstrated that the binding of maltotriose to the protein is exothermic and tight, whereas no thermal effect was observed upon addition of maltose at three temperatures. Therefore, PfuMBP evidently is designed to bind oligosaccharides composed of three or more glucopyranose units.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Oligossacarídeos/metabolismo , Pyrococcus furiosus/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Calorimetria , Proteínas de Transporte/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/química , Ligação de Hidrogênio , Maltose/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Periplásmicas de Ligação , Ligação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Propriedades de Superfície , Termodinâmica , Trissacarídeos/metabolismo
7.
Protein Eng ; 14(12): 993-1000, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11809930

RESUMO

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.


Assuntos
Endopeptidases/química , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Endopeptidases/genética , Endopeptidases/fisiologia , Estabilidade Enzimática , Cinética , Dados de Sequência Molecular , Mutação , Engenharia de Proteínas , Alinhamento de Sequência
8.
Acta Crystallogr D Biol Crystallogr ; 56(Pt 12): 1676-9, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11092944

RESUMO

A recombinant form of Yersinia pestis YopM with a C-terminal polyhistidine affinity tag has been overproduced in Escherichia coli, purified to homogeneity and crystallized using the hanging-drop vapor-diffusion technique. Several different crystal forms were obtained. The most suitable crystals for X-ray diffraction belonged to space groups P4(2)22 (unit-cell parameters a = 109.36, b = 109.36, c = 101.50 A) and C222(1) (unit-cell parameters a = 71.73, b = 121. 85, c = 189.79 A). With a synchrotron-radiation source, these crystals diffracted to 2.4 and 1.9 A resolution, respectively.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Yersinia pestis/química , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Cristalografia por Raios X , Peste/microbiologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Virulência
9.
Protein Expr Purif ; 20(2): 186-95, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11049743

RESUMO

Interleukin-13 (IL-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. Recent studies underscore its role in several diseases, including asthma and cancer. Solution studies of IL-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. A synthetic gene encoding human IL-13 (hIL-13) was inserted into the pMAL-c2 vector with a cleavage site for the tobacco etch virus (TEV) protease. Coexpression of the fusion protein and TEV protease led to in vivo cleavage, resulting in high levels of hIL-13 production. hIL-13, localized to inclusion bodies, was purified and refolded to yield approximately 2 mg per liter of bacteria grown in minimal media. Subsequent biochemical and biophysical analysis of both the unlabeled and (15)N-labeled protein revealed a bioactive helical monomer. In addition, the two disulfide bonds were unambiguously demonstrated to be Cys29-Cys57 and Cys45-Cys71 by a combined proteolytic digestion and mass spectrometric analysis.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Interleucina-3/isolamento & purificação , Interleucina-3/metabolismo , Proteínas de Transporte de Monossacarídeos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Renaturação Proteica , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Escherichia coli , Humanos , Interleucina-3/química , Interleucina-3/genética , Espectroscopia de Ressonância Magnética , Proteínas Ligantes de Maltose , Espectrometria de Massas , Desnaturação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
10.
Protein Expr Purif ; 19(2): 312-8, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10873547

RESUMO

Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease. A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector. The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector. In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines. Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer. Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins. When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process. Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate. This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted.


Assuntos
Endopeptidases/genética , Potyvirus/química , Proteínas Recombinantes de Fusão/metabolismo , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Vetores Genéticos , Proteínas Recombinantes de Fusão/genética , Solubilidade
11.
J Biol Chem ; 275(9): 6530-6, 2000 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-10692458

RESUMO

The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.


Assuntos
Ácidos Nucleicos Heteroduplexes/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Biotinilação , DNA/metabolismo , Estabilidade Enzimática , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico
12.
Protein Sci ; 8(8): 1668-74, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10452611

RESUMO

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Transporte de Monossacarídeos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Primers do DNA , Glutationa Transferase/química , Maltose/metabolismo , Proteínas Ligantes de Maltose , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Tiorredoxinas/química
13.
EMBO J ; 18(14): 3947-55, 1999 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10406799

RESUMO

We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar aspartic proteinase. In addition to the classical pepsin-like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant-specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N-terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant-specific domain with NK-lysin indicates that these two saposin-like structures are closely related, suggesting that all saposins and saposin-like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor-binding site involved in Golgi-mediated transport to vacuoles.


Assuntos
Ácido Aspártico Endopeptidases/química , Catepsinas/química , Precursores Enzimáticos/química , Hordeum/enzimologia , Vacúolos/enzimologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Transporte Biológico , Catepsinas/metabolismo , Cristalografia por Raios X , Dissulfetos/química , Elétrons , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Glicoproteínas/química , Complexo de Golgi/metabolismo , Hordeum/citologia , Hordeum/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteolipídeos/química , Surfactantes Pulmonares/química , Receptores de Superfície Celular/metabolismo , Saposinas , Homologia de Sequência de Aminoácidos
14.
Anal Biochem ; 262(2): 122-8, 1998 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-9750126

RESUMO

Site-specific, enzymatic biotinylation of recombinant proteins can be exploited to circumvent many problems associated with the use of biotinylating reagents in vitro and to overcome some of their inherent limitations. Additionally, biotinyl proteins can be purified to near-homogeneity in a single step under native conditions. Here we report that a biotin acceptor peptide (BAP) substrate for Escherichia coli biotin holoenzyme synthetase (BirA) can be used to label recombinant proteins with biotin in Spodoptera frugiperda (Sf9) cells, and we describe a collection of baculovirus transfer vectors specifically designed for this purpose. These BioBac vectors will greatly expand the range of proteins to which this technology can be applied.


Assuntos
Biotina/análogos & derivados , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Spodoptera/química , Acetil-CoA Carboxilase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Biotinilação , Proteínas de Transporte/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II , Vetores Genéticos/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/análise , Sulfurtransferases
15.
J Biol Chem ; 273(27): 17109-14, 1998 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-9642277

RESUMO

The multifunctional signal transducer and activator of transcription (STAT) proteins relay signals from the cell membrane to the nucleus in response to cytokines and growth factors. STAT4 becomes activated when cells are treated with interleukin-12, a key cytokine regulator of cell-mediated immunity. Upon activation, dimers of STAT4 bind cooperatively to tandem interferon-gamma activation sequences (GAS elements) near the interferon-gamma gene and stimulate its transcription. The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-gamma transcription in response to interleukin-12. We have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to homogeneity for structural studies. The circular dichroism spectrum of hSTAT4(1-124) indicates that it has a well ordered conformation in solution. The translational diffusion constant of hSTAT4(1-124) was determined by nuclear magnetic resonance methods and found to be consistent with that of a dimer. The rotational correlation time (tauc) of hSTAT4(1-124) was estimated from 15N relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric protein. These results, together with the number of signals observed in the two-dimensional 1H-15N heteronuclear single quantum coherence spectrum of uniformly 15N-labeled protein, indicate that hSTAT4(1-124) forms a stable, symmetric homodimer in solution. Cooperativity in native STAT4 probably results from a similar or identical interaction between the amino-terminal domains of adjacent dimers bound to DNA.


Assuntos
Proteínas de Ligação a DNA/química , Transativadores/química , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Fator de Transcrição STAT4 , Transativadores/genética , Transativadores/isolamento & purificação
16.
Protein Expr Purif ; 11(3): 233-40, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9425626

RESUMO

The native Ras farnesyltransferase heterodimer (alpha beta) and a heterodimer with a truncated alpha subunit (alpha' beta) were overproduced at a high level and in a soluble form in Escherichia coli. The alpha, alpha', and beta subunits were synthesized from individual plasmid vectors under the control of bacteriophage T7 promoters. Although each subunit could be expressed at a high level by itself, when either the alpha or alpha' and the beta plasmid were present in cells at the same time, the alpha and alpha' subunits were preferentially expressed to such a degree that little or none of the beta subunit accumulated. A satisfactory balance between both combinations of subunits (alpha beta and alpha' beta) was achieved by making incremental adjustments in the copy number of the beta-encoding plasmid. As the copy number of the beta plasmid increased, so did the ratio of beta:alpha or beta:alpha', but there was little difference in the total amount of recombinant protein (alpha + beta or alpha' + beta) that was produced. This may be a generally useful method for balancing the production of two recombinant polypeptides in E. coli. A noteworthy advantage of this approach is that it can be undertaken without first determining the cause of the imbalance.


Assuntos
Alquil e Aril Transferases/biossíntese , Alquil e Aril Transferases/química , Clonagem Molecular/métodos , Alquil e Aril Transferases/isolamento & purificação , Sequência de Aminoácidos , Bacteriófago T7 , Sequência de Bases , Primers do DNA , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Vetores Genéticos , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligopeptídeos , Peptídeos , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
17.
J Biomol NMR ; 8(2): 184-92, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8914274

RESUMO

A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any 15N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.


Assuntos
Aminoácidos/biossíntese , Proteínas de Bactérias/biossíntese , Escherichia coli/genética , Marcação por Isótopo/métodos , Bacteriólise , Elementos de DNA Transponíveis , Escherichia coli/metabolismo , Marcadores Genéticos , Genótipo , Mutação , Isótopos de Nitrogênio , Transdução Genética
18.
Drug Des Discov ; 13(3-4): 83-93, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8874046

RESUMO

The structure of the Ras-binding domain of human c-Raf-1 (residues 55 to 132) as determined in solution by NMR spectroscopy is presented. It consists of a five-stranded beta-sheet, a twelve residue alpha-helix, and an additional one-turn helix. The fold belongs to a known family whose members include ubiquitin and protein G. The surface of Raf55-132 that interacts with Ras has been identified by resonance perturbation mapping. The binding site is a spatially contiguous patch comprised of the two-N-terminal beta-strands, the loop between them, and the C-terminal end of the alpha-helix. A model of the Raf-Ras complex is presented, which was derived by analogy to the complex between protein G and a Fab fragment of IgG. In the model, edge beta-strands of each protein align in an antiparallel orientation, forming a unified beta-sheet, and side chains from both proteins are able to participate in ionic and hydrophobic interactions at the interface.


Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Proteínas ras/química , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-raf , Ubiquitinas/química , Proteínas ras/metabolismo
19.
Gene ; 169(1): 59-64, 1996 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-8635750

RESUMO

A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (MalE) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Biotina , Proteínas de Escherichia coli , Vetores Genéticos , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de Ligação , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Primers do DNA/química , Escherichia coli , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Proteínas Recombinantes/química , Sulfurtransferases/metabolismo
20.
Protein Expr Purif ; 6(6): 737-47, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8746625

RESUMO

A soluble, biologically active form of IL-2R alpha known as delta MST and consisting of the 178 N-terminal amino acid residues of the mature protein was directly expressed in the cytoplasm and the periplasm of Escherichia coli. Because it was not glycosylated, the E. coli protein was substantially less heterogeneous than delta MST expressed in insect cells. Nevertheless, it manifested equivalent biological activity in an IL-2 binding assay. The level of active delta MST production was higher when the protein was expressed in secretable form with a bacterial signal peptide than when it was produced in the cytoplasm, probably because the oxidizing environment and the presence of disulfide isomerases in the periplasm facilitated the correct folding of delta MST.


Assuntos
Receptores de Interleucina-2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Citoplasma/química , DNA Recombinante/genética , Dissulfetos/química , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Humanos , Interleucina-2/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Conformação Proteica , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-2/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Spodoptera
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