Membrane Protein Production for Structural Analysis

Mus‐Veteau, Isabelle; Demange, Pascal; Zito, Francesca

doi:10.1007/978-1-4939-0662-8_1

Cited by 4 publications

(3 citation statements)

References 194 publications

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“…A nonionic detergent n-Dodecyl-B-D-Maltopyranoside (DDM), being a “standard” class of its own as it is one of the most commonly used compounds in membrane protein purification. A 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG), another nonionic detergent with branched-chains, which is reported to form more stable protein-detergent complex and stabilizes the protein itself [ 63 ]. From the second zwitterionic class, Fos-Choline-12 (FC12), with strong extraction capabilities and n-Dodecyl-N,N-Dimethylamine-N-Oxide (LDAO) as one of the most successful of the zwitterionic detergents [ 64 ] were selected, see Table 3 .…”

Section: Methodsmentioning

confidence: 99%

Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

et al. 2015

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Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

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Section: Methodsmentioning

confidence: 99%

Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

et al. 2015

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“…Membrane proteins continue to be a challenge to work with. This is especially true when trying to produce them for structural characterization, which often necessitates over‐expressing and producing them recombinantly using model microbial systems, namely E. coli and yeast‐based systems (Bian et al, 2012; Mus, 2014; O'Malley et al, 2006; Pines & Inouye, 1999; Routledge et al, 2016; Saida et al, 2006). Even under successful conditions, there are still concerns about structural integrity and post‐translational processing that must be considered before attempting to interpret structural data and deduce important insights, which might otherwise be questionable (Haines & Irvine, 2003; Oueslati et al, 2010; Snider & Omary, 2014).…”

Section: Introductionmentioning

confidence: 99%

Predicted N‐terminal N‐linked glycosylation sites may underlie membrane protein expression patterns in Saccharomyces cerevisiae

Karki

Rimal

Rieth

2021

Yeast

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N‐linked glycosylation is one type of posttranslational modification that proteins undergo during expression. The following describes the effects of N‐linked glycosylation on high‐level membrane protein expression in yeast with an emphasis on Saccharomyces cerevisiae. N‐linked glycosylation is highlighted here as an important consideration when preparing membrane protein gene constructs for expression in S. cerevisiae, which continues to be used as a workhorse in both research and industrial applications. Non‐native N‐linked glycosylation commonly occurs during the heterologous expression of mammalian proteins in many yeast species which can have important immunological consequences when used in the production of biotherapeutic proteins or peptides. Further, non‐native N‐linked glycosylation can lead to improper protein folding and premature degradation, which can impede high‐level expression yields and hinder downstream analysis. Multiple strategies are presented in this article, which suggest different methods that can be implemented to circumvent the unwanted consequences of N‐linked glycosylation during the expression process. These considerations may have long‐term benefits for high‐level protein production in S. cerevisiae across a broad spectrum of expression targets with special emphasis placed on G‐protein coupled receptors, one of the largest families of membrane proteins.

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“…No entanto, ainda que simples do ponto de vista de preparo para a simulação, essa técnica é muito dispendiosa computacionalmente, por requerer longo, por vezes impraticável, tempo de trajetória e amostragem da flutuação da estrutura ligantes, assim como do receptor, de tal forma a observar eventos de ligação e dissociação do sítio de interação e obter uma correta amostragem. [74] Por outro lado, uma grande vantagem da técnica é permitir acompanhar o caminho de acesso à cavidade em detalhes, bem como a influência de outras moléculas do ligante e, até mesmo, a interação concomitante de um ou mais ligantes ao mesmo sítio de interação.…”

Section: Docking Molecularunclassified

Ação de anestésicos gerais em canais iônicos

Hosoume¹

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Primeiramente, gostaria de agradecer muitíssimo à minha família, Eidy e Célia. Não há como expor em palavras o quanto vocês me ajudaram e ajudam durante toda a minha jornada, em todos os aspetos. Obrigada pelo apoio e atenção incondicional. À vocês tenho admiração e gratidão imensuráveis.Ao Jonathan por todo o companheirismo e carinho, por me escutar e me dar forças sempre que preciso. Obrigada por proporcionar momentos de descanso e descontração, além de sempre me motivar a fazer planos e lutar pelo que quero.Ao Dr. Werner Treptow, meu orientador, por todo suporte, paciência, orientação e tempo. Por sempre me receber bem, ainda que fosse por dúvidas bobas ou grandes questionamentos. Sua ajuda, orientação e disposição foram indispensáveis para a conclusão deste trabalho. Aprendi e tenho muito ainda o que aprender contigo. Devo grande parte do que sei hoje a esse grande profissional. Muito obrigada! Agradeço ao pessoal do LBTC (Laboratório de Biologia Teórica e Computacional) Caio, Letícia, Vinícius, Camila, Alessandra e Mônica, além do Danilo, do Leonardo e do Matheus, por todas as conversas, paciência e momentos de descontração. Por dividirem comigo todos os anseios da pesquisa. Obrigada por estarem dispostos a ajudar em tudo.A todos da biofísica por proporcionarem uma ótima convivência. A todos também do programa de pós-graduação, por sempre atenderem e estarem dispostos a dar auxílio necessário.Aos membros da banca por cederem tempo para leitura do trabalho e pela disposição para contribuir com trabalho.Agradeço também ao CNPq pelo apoio financeiro.

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Membrane Protein Production for Structural Analysis

Cited by 4 publications

References 194 publications

Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

Predicted N‐terminal N‐linked glycosylation sites may underlie membrane protein expression patterns in Saccharomyces cerevisiae

Ação de anestésicos gerais em canais iônicos

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