Name: identification of kinase-substrate pairs using high throughput screening
Uploaded: 2015-08-29T14:00:00
Description: Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

This work was supported by NSERC grant 386634. We would like to thank members of the Screaton Lab for helpful discussions.

1. Preparation of Reagents, Plates, and Cells
<ol>
	<li>Make 500 ml lysis buffer: 25 mM Tris pH 7.5, 150 mM NaCl, 50 mM NaF, 0.5 mM EDTA pH 8.0, 0.5% TritonX-100, 5 mM beta-glycerophosphate, 5% glycerol. Store at 4 &#176;C. Immediately prior to use, add 1 mM dithiothreitol (DTT), 1 mM phenylmethyl sulfonyl fluoride (PMSF), and 1 mM sodium vanadate. After this step, PMSF is not required in any rinse buffer.</li>
	<li>Make 20 ml of 10x Kinase Buffer: 200 mM Tris pH 7.5, 50 mM beta-glycerophosphate (FW 216), 2 mM sodium v...

<ol>
	<li>Meisenhelder, J., Hunter, T., van der Geer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphopeptide+mapping+and+identification+of+phosphorylation+sites.">Phosphopeptide mapping and identification of phosphorylation sites.</a> Curr Protoc Mol Biol. 18, Unit 18 19 (2001).</li><li>Doll, S., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+detection+and+assignment+of+protein+posttranslational+modifications.">Mass spectrometry-based detection and assignment of protein posttranslational modifications.</a> ACS chem. 10, 63-71 (2015).</li><li>Sharma, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultradeep+human+phosphoproteome+reveals+a+distinct+regulatory+nature+of+Tyr+and+Ser/Thr-based+signaling.">Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.</a> Cell rep. 8, 1583-1594 (2014).</li><li>Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+protein+function+by+multisite+phosphorylation--a+25+year+update.">The regulation of protein function by multisite phosphorylation--a 25 year update.</a> Trends Biochem Sci. 25, 596-601 (2000).</li><li>Walsh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Posttranslation Modification of Proteins: Expanding Nature's Inventory. , (2006).</li><li>Boersema, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+qualitative+and+quantitative+profiling+of+tyrosine+phosphorylation+using+a+combination+of+phosphopeptide+immunoaffinity+purification+and+stable+isotope+dimethyl+labeling.">In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.</a> Mol Cell Proteomics. 9, 84-99 (2010).</li><li>Hutti, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+determining+protein+kinase+phosphorylation+specificity.">A rapid method for determining protein kinase phosphorylation specificity.</a> Nat Methods. 1, 27-29 (2004).</li><li>Johnson, S. A., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinomics:+methods+for+deciphering+the+kinome.">Kinomics: methods for deciphering the kinome.</a> Nat Methods. 2, 17-25 (2005).</li><li>Pawson, T., Nash, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+cell+regulatory+systems+through+protein+interaction+domains.">Assembly of cell regulatory systems through protein interaction domains.</a> Science. 300, 445-452 (2003).</li><li>Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+yeast+protein+kinases+using+protein+chips.">Analysis of yeast protein kinases using protein chips.</a> Nat Genet. 26, 283-289 (2000).</li><li>Shah, K., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+approach+for+the+identification+of+direct+substrates+of+protein+kinases.">A chemical genetic approach for the identification of direct substrates of protein kinases.</a> Methods Mol Biol. 233, 253-271 (2003).</li><li>Zou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKIS:+computational+identification+of+protein+kinases+for+experimentally+discovered+protein+phosphorylation+sites.">PKIS: computational identification of protein kinases for experimentally discovered protein phosphorylation sites.</a> BMC bioinform. 14, 247 (2013).</li><li>Varjosalo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+active+and+kinase-deficient+kinome+collection+for+identification+of+kinases+regulating+hedgehog+signaling.">Application of active and kinase-deficient kinome collection for identification of kinases regulating hedgehog signaling.</a> Cell. 133, 537-548 (2008).</li><li>Jansson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+controls+CREB+activity+in+islet+cells+via+regulated+phosphorylation+of+TORC2.">Glucose controls CREB activity in islet cells via regulated phosphorylation of TORC2.</a> Proc Natl Acad Sci U S A. 105, 10161-10166 (2008).</li><li>Fu, A., Screaton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+kinomics+to+delineate+signaling+pathways:+control+of+CRTC2/TORC2+by+the+AMPK+family.">Using kinomics to delineate signaling pathways: control of CRTC2/TORC2 by the AMPK family.</a> Cell Cycle. 7, 3823-3828 (2008).</li><li>Abu-Thuraia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axl+phosphorylates+elmo+scaffold+proteins+to+promote+rac+activation+and+cell+invasion.">Axl phosphorylates elmo scaffold proteins to promote rac activation and cell invasion.</a> Mol Cell Biol. 35, 76-87 (2015).</li><li>Manning, G., Whyte, D. B., Martinez, R., Hunter, T., Sudarsanam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+complement+of+the+human+genome.">The protein kinase complement of the human genome.</a> Science. 298, 1912-1934 (2002).</li></ol>

Since the original publications describing the approach 14,15, the original library of 180 GST-kinases has been expanded to 420 members, or ~80% of the human protein kinome. With the expanded library, the protocol as described takes 4-5 days and then 1-4 days to develop films (as deemed necessary), which could be shortened by use of phosphorimaging and digital signal enhancement. There are several key steps where care must be taken (See Figure 1 for overview of protocol). First, is the health ...

Protein post-translational modifications (PTMs) are essential for intracellular communication. Perhaps the best studied of all PTMs is phosphorylation, catalyzed by protein kinases, which regulate a myriad of protein functions, including their biochemical activity, subcellular localization, conformation, and stability. The identification of phosphorylation sites on target proteins can be accomplished by tryptic phosphopeptide mapping or by now-standard proteomic techniques using samples enriched for phosphorylated peptides 1,2. While three quarters of the expressed proteome are expected to be phosphorylated 3 and an identified 200,000 phosphorylation sites 5, with estimates up to 1 million 6, many of these have no assigned biology, signaling pathway, or protein kinase.
While identification of phosphorylated sites is relatively straightforward, a comparatively greater challenge is to identify the cognate kinase(s) that targets these sites, a process we refer to as mapping kinase:substrate pairs. Several approaches for identifying kinase:substrate pairs have been described, either starting with a kinase of interest and looking for its substrates or starting with a substrate of interest and attempting to find a modifying kinase experimentally 7-11 or computationally 12. To identify kinases for a known phosphorylated substrate, bioinformatics can be used to identify proteins that contain a short conserved sequence of amino acids flanking the phosphorylated residue (the consensus site), as well as identifying kinases that form a precipitable complex with the substrate. However, these approaches are time-consuming and often do not meet with success.
We developed a systematic functional approach to rapidly identify kinases that can phosphorylate a given substrate 13. The screen assay produces excellent specificity, with very clear selection for potential cognate kinases. Given the centrality of phosphorylation to biological signaling, the screen is useful for discovery in virtually all cell signaling pathways 14-16. The screen involves performing a large-scale kinase assay with a library of human protein kinases. The kinases have been tagged with bacterial glutathione S-transferase (GST) protein and are purified from mammalian cell extracts, which means that the recombinant enzymes &#8212; unlike those prepared from bacteria &#8212; are generated in the presence of the upstream protein kinases often required for the recombinant enzymes to have activity in vitro. Indeed, while serine, threonine, and tyrosine kinase activity required for downstream kinase activation are present in yeast 10, the yeast genome encodes 122 protein kinases, indicating that the mammalian kinome, with over 500 genes 17, has become significantly more complex in order to regulate the processes unique to higher order organisms. Moreover, the effect of different stimuli relevant to cell biology and human disease (such as small molecules, growth factors, hormones, etc.) can be used to 14,15modulate kinase activity in an appropriate context.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Representative results from a screen are shown in Figure 2. 180 kinases were screened using a GST-tagged peptide substrate corresponding to aa 268-283 from CRTC2 as well as the classic kinase assay substrate myelin basic protein (MBP). Only two kinases, MARK2 and the highly related kinase MARK3 phosphorylated the CRTC2 peptide. MBP is included as an internal control in all assays, as it contains many phosphorylatable residues and runs at 18 kDa, toward the bottom of the gel. This allows for an interpreta...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

Research

JoVE Journal

Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...