cloning of solute carriers  &#40;slcs&#41; into phtbv1.1 bacmid for high-throughput protein expression 

Estratégias para expressar e purificar transportadores de soluto

Membrane proteins have long been targets for researchers and pharmaceutical industries alike. Of these, the solute carriers (SLCs) are a family of over 400 secondary transporter genes encoded within the human genome1. These transporters are involved in the import and export of numerous molecules, including ions2, neurotransmitters3, lipids4,5,6,7, amino acids8, nutrients9,10,11, and pharmaceuticals12. With such a breadth of substrates, these proteins are also implicated in a range of pathophysiologies through the transport of toxins13, transport of and inhibition by drugs of abuse14,15, or deleterious mutations16. Bacterial homologs have served as prototypes for the fundamental transport mechanism of several SLC families17,18,19,20,21,22,23,24,25. In contrast to human proteins, prokaryotic orthologs are often better expressed in the well-understood Escherichia coli expression system26,27 and are more stable in the smaller detergents which yield well-ordered crystals for X-ray crystallography28. However, sequence and functional differences complicate the use of these distantly-related proteins for drug discovery29,30. Consequently, direct study of the human protein is often needed to decipher the mechanism of action of drugs targeting SLCs31,32,33,34,35. While the recent advances in Cryo-electron Microscopy (Cryo-EM) have enabled structural characterization of SLCs in more native-like conditions36,37, difficulty in expressing and purifying these proteins remains a challenge for developing targeted therapeutics and diagnostics.
To alleviate this challenge, the RESOLUTE consortium (re-solute.eu) has developed resources and protocols for the large-scale expression and purification of human SLC-family proteins38. Starting with codon-optimized genes, we have developed methods for the high-throughput cloning and screening of SLC constructs. These methods were systematically applied to the whole family of SLCs, the genes were cloned into the BacMam viral expression system, and the protein expression was tested in human cell lines39 based on previously described methods for high-throughput cloning and expression testing40. In summary, the SLC gene is cloned from the pDONR221 plasmid into a pHTBV1.1 vector. This construct is subsequently used to transpose the gene of interest into a bacmid vector for transfecting insect cells, which includes a cytomegalovirus promoter and enhancer elements for expression in mammalian cells. The resulting baculovirus can be used to transduce mammalian cells for the expression of the target SLC protein.
We further developed standardized methods for large-scale expression and stable purification of selected SLCs (Figure 1). This protocol includes multiple checkpoints to facilitate effective troubleshooting and minimize variability between experiments. Notably, routine monitoring of protein expression and localization, as well as small-scale optimization of purification conditions for individual targets, were aided by Strep and Green Fluorescent Protein (GFP) tags41,42.
Ultimately, these chemically pure and structurally homogeneous protein samples can be used for structural determination by X-ray crystallography or Cryo-Electron Microscopy (Cryo-EM), biochemical target-engagement assays, immunization for binder generation, and cell-free functional studies via reconstitution into chemically defined liposomes.

Autor em destaque: Expressão e purificação de transportadores de soluto humano usando genes otimizados para códons 

Expressão de alto rendimento e purificação de carreadores de solutos humanos para estudos estruturais e bioquímicos

Solute carriers, or SLCs, are responsible for uptaking nutrients and are involved in human diseases. However, there are few drugs targeting SLCs because they're poorly studied. We developed efficient approaches to clone, express, and purify SLCs.Our workflow yields pure proteins, allowing biochemical and structural studies, and ultimately provides insights for drug discovery campaigns. With the numerous roles in biological processes, disease and drug delivery, SLCs have increasingly received the attention and become attractive therapeutical targets. The private, public ReSOLUTE IMI projects generated open access resources and data to enable novel discoveries in SLC function, physiology, pathology, and therapeutic targeting.Traditionally, SLCs were challenging to study as they are integral membrane proteins with various substrates and diverse transport mechanisms. Recently, Cryo-EM, the BacMam expression system, and new solubilization reagents have led to a step change in their characterization. Pure protein samples are needed for biochemical and structural studies.In human solute carriers often have a poor yield when over expressed and unstable when purified. Therefore, in the past, each target required years to develop individual structures to produce the protein sample for downstream applications. Our method is optimized for the parallel and cost-efficient study of multiple targets or constructs of a single target.Therefore, it balances experimental flexibility, cost, time, and lab resources carefully. Plus, we offer open access to all our protocols and resources in this article and in the ReSOLUTE web portal.

Watch this Scientific Journal Video about Cloning of Solute Carriers  SLCs into pHTBV1.1 Bacmid for High-Throughput Protein Expression at JoVE.com

Cloning of Solute Carriers  (SLCs) into pHTBV1.1 Bacmid for High-Throughput Protein Expression   

Clonagem de Transportadores de Solutos (SLCs) em pHTBV1.1 Bacmid para Expressão de Proteína de Alto Rendimento 

Para começar, adicione 150 nanogramas de carregador de soluto, ou clone SLC, e 100 nanogramas do vetor em uma placa de 96 poços. Fazer o volume de reação para 8 microlitros com solução tris 10 milimolares pH 8. Em seguida, adicione 2 microlitros de mistura enzimática recombinante e incube por uma hora à temperatura ambiente.Após a incubação, adicionar 1 microlitro de proteinase K e incubar por 30 minutos a 37 graus Celsius. Adicione 4 microlitros de mistura de reação a 50 microlitros de células de E.coli Mach1 quimicamente competentes e transforme as células usando o método de choque térmico. Após a transformação, colocar a suspensão bacteriana em uma placa de ágar LB.Identificar colônias que abrigam o vetor pHTBV1.1 com a inserção do gene SLC usando primers apropriados e protocolos padrão para PCR de colônias.

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Research

JoVE Journal

Biochemistry

Cloning of Solute Carriers  &#40;SLCs&#41; into pHTBV1.1 Bacmid for High-Throughput Protein Expression 

175 visualizações.  University of Oxford. Para começar, adicione 150 nanogramas de carregador de soluto, ou clone SLC, e 100 nanogramas do vetor em uma placa de 96 poços. Fazer o volume de reação para 8 microlitros com solução tris 10 milimolares pH 8. Em seguida, adicione 2 microlitros de mistura enzimática recombinante e incube por uma hora à temperatura ambiente.Após a incubação, adicionar 1 microlitro de proteinase K e incubar por 30 minutos a 37 graus Celsius. Adicione 4 microlitros de mistura de reação a 50 ...