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

אסטרטגיות לביטוי וטיהור מובילי מובילי מומסים

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.

מחבר זרקור: ביטוי וטיהור של מובילי נשאי מומסים אנושיים באמצעות גנים ממוטבים של קודון 

ביטוי וטיהור בתפוקה גבוהה של נשאי מומסים אנושיים למחקרים מבניים וביוכימיים

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   

שיבוט נשאי מומסים (SLC) לתוך pHTBV1.1 Bacmid לביטוי חלבון בתפוקה גבוהה 

כדי להתחיל, הוסף 150 ננוגרם של נשא מומס, או שיבוט SLC, ו -100 ננוגרם של הווקטור בלוח 96 בארות. הפוך את נפח התגובה ל 8 מיקרוליטר עם תמיסת טריס 10 מילימולרית pH 8. לאחר מכן מוסיפים 2 מיקרוליטר של תערובת אנזימים רקומביננטיים ודגרים במשך שעה בטמפרטורת החדר.לאחר הדגירה, להוסיף 1 מיקרוליטר של proteinase K לדגור במשך 30 דקות ב 37 מעלות צלזיוס. הוסף 4 מיקרוליטר של תערובת תגובה ל 50 מיקרוליטר של תאי E.coli Mach1 מוכשרים כימית, ולהפוך את התאים באמצעות שיטת הלם חום. לאחר הטרנספורמציה, לוחצים את התרחיף החיידקי על צלחת אגר LB.זהה מושבות המכילות את וקטור pHTBV1.1 עם החדרת הגן SLC באמצעות פריימרים מתאימים ופרוטוקולים סטנדרטיים עבור PCR מושבה.

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Biochemistry

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

176 Views.  University of Oxford. כדי להתחיל, הוסף 150 ננוגרם של נשא מומס, או שיבוט SLC, ו -100 ננוגרם של הווקטור בלוח 96 בארות. הפוך את נפח התגובה ל 8 מיקרוליטר עם תמיסת טריס 10 מילימולרית pH 8. לאחר מכן מוסיפים 2 מיקרוליטר של תערובת אנזימים רקומביננטיים ודגרים במשך שעה בטמפרטורת החדר.לאחר הדגירה, להוסיף 1 מיקרוליט?...

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

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

To begin, add 150 nanograms of solute-carrier, or SLC clone, and 100 nanograms of the vector in a 96-well plate. Make the reaction volume to 8 microliters with 10 millimolar tris solution pH 8. Then add 2 microliters of recombinant enzyme mix and incubate for one hour at room temperature.After incubation, add 1 microliter of proteinase K and incubate for 30 minutes at 37 degrees Celsius. Add 4 microliters of reaction mixture to 50 microliters of chemically competent E.coli Mach1 cells, and transform the cells using the heat shock method. After transformation, plate the bacterial suspension onto an LB agar plate.Identify colonies harboring the pHTBV1.1 vector with the SLC gene insert using appropriate primers and standard protocols for colony PCR.

Purification of Human Solute Carriers  &#40;SLCs&#41; for Developing SLC Targeting Therapies

Purification of Human Solute Carriers  (SLCs) for Developing SLC Targeting Therapies  

Begin by thawing the frozen solute carriers or SLC cell pellets in a water bath at room temperature. Prepare solubilization buffer by combining 135 milliliters of base buffer and three protease inhibitor cocktail tablets. Once the tablets are dissolved, resuspend the pellet in the solubilization buffer.Then add deaminase and transfer the suspension into an ice cooled down homogenizer. Homogenize the solution by moving the plunger up and down approximately 20 times, keeping the homogenizer on ice. Next, add detergent stock solution to 1%final concentration.Transfer the solubilization mixture to a 50 milliliter conical tube and rotate slowly at four degrees Celsius. After one hour, centrifuge the mixture, and collect the supernatant. Equilibrate a four to six milliliter bed volume of Strep-Tactin resin with the base buffer.Then add equilibrated resin to the solubilized supernatant, and rotate for two hours at four degrees Celsius. Pour the solution onto a gravity flow column and allow the solution to flow through. Wash the resin with 30 times the bed volume of Strep Wash Buffer in three equal steps.Next, add three to five milliliters of illusion buffer and incubate for 15 minutes before collecting the elute. Measure protein concentration by UV absorbent spectroscopy. Combine the desired illusion fractions, and add 3C protease.Rotate the tube slowly overnight at four degrees Celsius. The next day, equilibrate two to four milliliters of bed volume of cobalt metal affinity resin with SEC buffer. Add the equilibrated cobalt metal affinity resin to the overnight 3C reaction mixture, and rotate at four degrees Celsius.After one hour, pour the solution into a gravity flow column and collect the flow through. Concentrate the flow through in a 100 kilodalton cutoff centrifugal filter by spinning at 3000g at four degrees Celsius. Equilibrated dextran argos based SEC column with SEC buffer.Inject the sample into the sample loop and run the SEC program with a flow rate, such that column pressure is below the column manufacturer's specifications. Using a fraction collector, collect 300 microliter fractions over the entire SEC run. After pooling peak fractions, measure the absorbance at 280 nanometers.Then concentrate the samples to the required volume in a 100 kilodalton cutoff filter. Initial small scale SLC protein expression was analyzed by SDS page electrophoresis. Fluorescence microscopy of A GFP tagged SLC, confirmed the protein localization, specific to the plasma membrane with significant intracellular accumulation.Chemically and structurally homogeneous protein yielded a single mono disperse A280 peak during size exclusion chromatography.