MultiBac: expanding the research toolbox for multiprotein complexes

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms

A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression

Patrimônio público escolar: da conscientização à preservação, um exercício de cidadania no ensino fundamental

Este trabalho apresenta o resultado de uma pesquisa-ação, a partir dos dados coletados e sua análise descritiva, frutos do Projeto de Intervenção intitulado “Patrimônio Público Escolar: da conscientização à preservação, um exercício de cidadania no Ensino Fundamental”, realizada em uma escola pública de Ensino Fundamental na cidade de Ipatinga, MG, para atender a uma demanda da escola no sentido de reelaborar o Projeto Político Pedagógico e adequá-lo às necessidades atuais. O objetivo principal foi conscientizar a comunidade escolar da necessidade de se reconhecer, respeitar e valorizar a escola como um bem público histórico e cultural, que deve ser preservado por todos. Os procedimentos metodológicos utilizados foram a pesquisa bibliográfica e o estudo de caso, com coleta de dados através de questionário. A implementação aconteceu no período de fevereiro a maio/2016, envolvendo 72 (setenta e dois) funcionários e 850 (oitocentos e cinqüenta) alunos. A análise dos resultados revelou que a depredação do patrimônio público escolar ocorre mais nas turmas de 7º, 8º e 9º ano. Muitos destes alunos vivem em situação de vulnerabilidade social.info:eu-repo/semantics/publishedVersio

Synthesis of Gold Nano-structure and its Photoelectronic Function

Nagasaki Symposium on Nano-Dynamics 2008 (NSND2008) 平成20年1月29日(火)於長崎大学 Poster Presentatio

Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion

Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex

Physical Education which Deepens Strategical Recognition by Accepting the Other Senses of Value (2) : Through the Class of Basketball Using ""the Game-stop and Interview"

The purpose of this study is to help students acquision of the ability which this school aims at, through the physical education. The abilities are, ""receipting various senses of values"", ""self-expression and communication"" and""decision-making"". In this study, we carried an approach focusing ""the ability to think scientifically"", ""determination and judgement"" and ""thought and judgement"". We carried out the method of ""the Game-stop and Interview"" for the practical study. As a result, the students' ability of offense and defense in a game was improved and has become much better than last academic year. This result proved that the method of ""the Game-stop and Interview"" was effective to improve the students' recognition for tactics