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1.
Acta Crystallogr F Struct Biol Commun ; 79(Pt 4): 82-86, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-36995122

RESUMO

CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Šresolution with the domain-swapping hinge loop (residues 379-387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer-dimer interconversion and for the optimization of conjugation.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Vacinas Conjugadas/química , Cristalografia por Raios X , Proteínas de Bactérias/química , Polissacarídeos , Desenvolvimento de Vacinas
2.
J Pharm Sci ; 107(7): 1806-1819, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29526446

RESUMO

Cross-reacting material 197 (CRM197), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM197 molecules demonstrate that recombinant CRM197's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM197 expressed in alternative sources have the potential to replace CRM197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM197 carrier proteins for use in various conjugate vaccines.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Animais , Anticorpos/imunologia , Proteínas de Bactérias/imunologia , Corynebacterium diphtheriae/genética , Escherichia coli/genética , Expressão Gênica , Humanos , Agregados Proteicos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Pseudomonas fluorescens/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Solubilidade , Vacinas Conjugadas/química , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologia
3.
Proteins ; 80(12): 2810-7, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23011838

RESUMO

Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains.


Assuntos
Plasmodium falciparum/química , Subtilisinas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Soluções
4.
Protein Expr Purif ; 52(2): 280-5, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17126029

RESUMO

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.


Assuntos
Resposta ao Choque Térmico , Proteínas Recombinantes/metabolismo , Cristalização , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
5.
J Struct Funct Genomics ; 6(2-3): 63-70, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16211501

RESUMO

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Modelos Moleculares , Mycoplasma genitalium/genética , Mycoplasma pneumoniae/genética , Dobramento de Proteína , Proteômica/métodos , Clonagem Molecular , Cristalização
6.
J Struct Funct Genomics ; 6(2-3): 177-82, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16211516

RESUMO

One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC). Our method is a combination of an 'artificial chaperone-assisted refolding' method previously proposed and affinity chromatography to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit of protein purification. It can be easily automated and formatted for high-throughput process.


Assuntos
Corpos de Inclusão/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas/isolamento & purificação , Proteômica/métodos , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Proteínas/metabolismo
7.
J Bacteriol ; 187(12): 4238-44, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15937186

RESUMO

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Fosfatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
9.
J Biol Chem ; 280(18): 18326-35, 2005 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-15753098

RESUMO

We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many alpha/beta barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr(21) and Phe(138). Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity.


Assuntos
Proteínas Arqueais/química , Pentosiltransferases/química , Thermoplasma/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/isolamento & purificação , Sítios de Ligação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Pentosiltransferases/isolamento & purificação
10.
J Struct Funct Genomics ; 5(1-2): 23-7, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15263840

RESUMO

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.


Assuntos
Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Automação , Expressão Gênica , Immunoblotting , Proteômica , Proteínas Recombinantes/biossíntese , Robótica , Solubilidade
11.
J Biol Chem ; 277(28): 24851-4, 2002 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-12034704

RESUMO

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.


Assuntos
Fatores de Coagulação Sanguínea , Fosfolipídeos/química , Proteína C/metabolismo , Receptores de Superfície Celular/química , Animais , Células CHO , Cricetinae , Cristalografia por Raios X , Modelos Moleculares , Mutagênese , Fosfolipídeos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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