Asunto(s)
Arabidopsis/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/metabolismo , Marcación de Gen/métodos , Mutagénesis Insercional/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endodesoxirribonucleasas/genética , Edición Génica , Células Germinativas de las Plantas/metabolismo , Recombinación Homóloga , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genéticaRESUMEN
Proper protein folding is a prerequisite for protein stability and enzymatic activity. Although directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (ß/α)8 barrel fold, which is found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (ß/α)8 barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants, and then combined them to assemble the final library (10¹4 DNA sequences). The resulting library was analyzed by using the in vitro protease assay and an in vivo GFP-folding assay; it contains â¼10¹² soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (ß/α)8-barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput mRNA display method with selection for folding. The resulting (ß/α)8 barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (ß/α)8 fold, and to isolate novel enzymes.
Asunto(s)
Biblioteca de Péptidos , Pliegue de Proteína , Estructura Secundaria de Proteína , Clonación Molecular , Activación Enzimática , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/metabolismo , Thermotoga maritima/enzimologíaRESUMEN
An artificial RNA ligase specific to RNA with a 5'-triphosphate (PPP-RNA) exhibits broad sequence specificity on model substrates and secondary siRNAs with direct applications in the identification of PPP-RNAs through sequencing.
Asunto(s)
ARN Ligasa (ATP)/química , ARN/química , ARN/genética , ARN Ligasa (ATP)/genética , ARN Interferente Pequeño/genética , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
In the past decade, in vitro evolution techniques have been used to improve the performance or alter the activity of a number of different enzymes and have generated enzymes de novo. In this review, we provide an overview of the available in vitro methods, their application, and some general considerations for enzyme engineering in vitro. We discuss the advantages of in vitro over in vivo approaches and focus on ribosome display, mRNA display, DNA display technologies, and in vitro compartmentalization (IVC) methods. This review aims to help researchers determine which approach is best suited for their own experimental needs and to highlight that in vitro methods offer a promising route for enzyme engineering.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Ingeniería de Proteínas/métodos , Evolución Molecular Dirigida , Enzimas/genética , Biblioteca de GenesRESUMEN
Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.
Asunto(s)
Catálisis , Ingeniería de Proteínas/métodos , ARN Ligasa (ATP)/química , Alanina/química , Secuencia de Aminoácidos , Dominio Catalítico , Evolución Molecular Dirigida/métodos , Enzimas/química , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Metales/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Pliegue de ProteínaRESUMEN
Stem cell homing and breast cancer metastasis are orchestrated by the chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4. Here, we report the nuclear magnetic resonance structure of a constitutively dimeric SDF-1 in complex with a CXCR4 fragment that contains three sulfotyrosine residues important for a high-affinity ligand-receptor interaction. CXCR4 bridged the SDF-1 dimer interface so that sulfotyrosines sTyr7 and sTyr12 of CXCR4 occupied positively charged clefts on opposing chemokine subunits. Dimeric SDF-1 induced intracellular Ca2+ mobilization but had no chemotactic activity; instead, it prevented native SDF-1-induced chemotaxis, suggesting that it acted as a potent partial agonist. Our work elucidates the structural basis for sulfotyrosine recognition in the chemokine-receptor interaction and suggests a strategy for CXCR4-targeted drug development.
Asunto(s)
Quimiocina CXCL12/química , Modelos Moleculares , Receptores CXCR4/química , Tirosina/análogos & derivados , Secuencia de Aminoácidos , Calcio/metabolismo , Línea Celular , Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito , Dimerización , Humanos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Receptores CXCR4/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMEN
We have applied an efficient solid-phase protein refolding method to the milligram scale production of natively folded recombinant chemokine proteins. Chemokines are intensely studied proteins because of their roles in immune system regulation, response to inflammation, fetal development, and numerous disease states including, but not limited to, HIV-1/AIDS, cancer metastasis, Crohn's disease, asthma and arthritis. Many investigators use recombinant chemokines for research purposes, however these proteins partition almost exclusively to the inclusion body fraction when produced in Escherichia coli. A major hurdle is to correctly refold the chemokine and oxidize the two highly conserved disulfide bonds found in nearly all chemokines. Conventional methods for oxidation and refolding by dialysis or extreme dilution are effective but slow and yield large volumes of dilute chemokine. Here we use an on-column approach for rapid refolding and oxidation of four chemokines, CXCL12/SDF-1alpha (stromal cell-derived factor-1alpha), CCL5/RANTES, XCL1/lymphotactin, and CX3CL1/fractalkine. NMR spectra of SDF-1alpha, RANTES, lymphotactin, and fractalkine indicate these chemokines adopt native structures. On-column refolded SDF-1alpha is fully active in an intracellular calcium flux assay. Our success with multiple SDF-1alpha mutants and members of all four chemokine subfamilies suggests that on-column refolding is a robust method for preparative-scale production of recombinant chemokine proteins.