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1.
J Struct Biol ; 203(2): 71-80, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29545204

RESUMO

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.


Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células Sf9
2.
Sci Rep ; 8(1): 4060, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497092

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

3.
G3 (Bethesda) ; 8(1): 79-89, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29118030

RESUMO

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Transposases/genética , Sequência de Bases , Clonagem Molecular/métodos , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Poliadenilação , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transposases/metabolismo
4.
Sci Rep ; 7(1): 1872, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28500343

RESUMO

The quantity of milk and milk fat and proteins are particularly important traits in dairy livestock. However, little is known about the regions of the genome that influence these traits in goats. We conducted a genome wide association study in French goats and identified 109 regions associated with dairy traits. For a major region on chromosome 14 closely associated with fat content, the Diacylglycerol O-Acyltransferase 1 (DGAT1) gene turned out to be a functional and positional candidate gene. The caprine reference sequence of this gene was completed and 29 polymorphisms were found in the gene sequence, including two novel exonic mutations: R251L and R396W, leading to substitutions in the protein sequence. The R251L mutation was found in the Saanen breed at a frequency of 3.5% and the R396W mutation both in the Saanen and Alpine breeds at a frequencies of 13% and 7% respectively. The R396W mutation explained 46% of the genetic variance of the trait, and the R251L mutation 6%. Both mutations were associated with a notable decrease in milk fat content. Their causality was then demonstrated by a functional test. These results provide new knowledge on the genetic basis of milk synthesis and will help improve the management of the French dairy goat breeding program.

5.
Science ; 326(5957): 1235-40, 2009 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-19965468

RESUMO

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.


Assuntos
Proteínas de Bactérias/análise , Genoma Bacteriano , Complexos Multiproteicos/análise , Mycoplasma pneumoniae/química , Mycoplasma pneumoniae/genética , Proteoma , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biologia Computacional , Espectrometria de Massas/métodos , Redes e Vias Metabólicas , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestrutura , Reconhecimento Automatizado de Padrão , Mapeamento de Interação de Proteínas , Biologia de Sistemas
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