Name: automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays
Uploaded: 2016-11-10T12:30:00
Description: Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

1. Peptide Serial Dilution
<ol>
	<li>Prepare assay buffer: Hanks buffered salt solution (HBSS) supplemented with 25 mM HEPES, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), pH 7.4.</li>
	<li>Use a bulk reagent dispenser to systematically add 5 &#181;l of assay buffer to each well of five 384-well low volume assay plates.
	<ol>
		<li>Use internal software to create a dispensing program for 5 &#181;l volume addition to every well of a 384-well plate as per manufacturer&#39;s instructions.</li>
		<li>Immerse disp...

<ol>
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R., 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=Bioactive+Contaminants+Leach+from+Disposable+Laboratory+Plasticware.">Bioactive Contaminants Leach from Disposable Laboratory Plasticware.</a> Science. 322 (5903), 917 (2008).</li><li>Belaiche, C., Holt, A., Saada, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonylphenol+ethoxylate+plastic+additives+inhibit+mitochondrial+respiratory+chain+complex+I.">Nonylphenol ethoxylate plastic additives inhibit mitochondrial respiratory chain complex I.</a> Clin Chem. 55 (10), 1883-1884 (2009).</li><li>Sackmann, E. 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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=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> J.Biomol.Screen. 14 (5), 452-459 (2009).</li><li>Turmel, M., Itkin, Z., Liu, D., Nie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Innovative+Way+to+Create+Assay+Ready+Plates+for+Concentration+Response+Testing+Using+Acoustic+Technology.">An Innovative Way to Create Assay Ready Plates for Concentration Response Testing Using Acoustic Technology.</a> J.Lab.Autom. 15 (4), 297-305 (2010).</li><li>Butler, R., 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=Use+of+the+site-specific+retargeting+jump-in+platform+cell+line+to+support+biologic+drug+discovery.">Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery.</a> J.Biomol.Screen. 20 (4), 528-535 (2015).</li><li>Panchal, S., Verma, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sodium+fluoride+in+maternal+and+offspring+rats+and+its+amelioration.">Effect of sodium fluoride in maternal and offspring rats and its amelioration.</a> Asian Pac.J.Reprod. 3 (1), 71-76 (2014).</li><li>Hanson, S. M., Ekins, S., Chodera, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+error+in+experimental+assays+using+the+bootstrap+principle:+understanding+discrepancies+between+assays+using+different+dispensing+technologies.">Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies.</a> J.Comput. Aided Mol.Des. 29 (12), 1073-1086 (2015).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+Contact-Free+Volume+Transfers+Minimize+Compound+Loss+in+Dose-Response+Experiments.">Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments.</a> J.Biomol. Screen. 15 (1), 86-94 (2010).</li><li>Chan, G. K. Y., Wilson, S., Schmidt, S., Moffat, J. 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This protocol describes the successful application of automated acoustic dispensing to serially dilute peptide samples over a concentration range of 3 x 106 requiring less than 1 &#181;l of sample. The major advantage of this method is to increase data quality through minimizing peptide adsorption to plasticware via reduced exposure of samples to experimental containers and plasticware (such as pipette tips) that are normally required for reagent transfer and mixing. While acoustic dispensing does not complete...

The GLP-1R is an established drug target in the treatment of type 2 diabetes2. The native peptide agonist for this receptor, GLP-1, has an in vivo half-life of 2-3 min3. The binding of GLP-1 to its G protein coupled target receptor results in the downstream production of the second messenger cAMP through native G protein coupling to the activation of adenylyl cyclase. Measurement of the accumulated cAMP provides a robust assay to monitor receptor activation and to screen for active GLP-1 analogues with preferred physicochemical properties. Such an assay requires the serial dilution of test samples to construct concentration-response curves, and this is particularly complicated when handing peptide samples. Potential errors from tip-based serial dilution preparation have been described previously1,4,5. Peptides will adsorb to plasticware, resulting in unreliable potency estimations. Peptide loss can be minimized through the inclusion of bovine serum albumin (BSA) in buffers and the use of siliconized plasticware, yet protein binding remains unpredictable. In particular, the variation in binding of GLP-1 to experimental containers has been described6. There is a further complication in that stabilization agents used in laboratory plasticware can leach from tips and microtiter plates into aqueous assay buffers and interfere with protein function7, 8. Therefore, methods to reduce exposure to plasticware are necessary to increase the accuracy of measurements.
Acoustic liquid dispensers focus a high-frequency acoustic signal onto the surface of a fluid sample, resulting in the ejection of precise nanoliter droplets into an adjacent assay plate9. The use of acoustic ejection is standard in the pharmaceutical industry for the preparation and screening of large synthetic compound libraries, and the technology has been well validated for small molecules10. To our knowledge, we are the first group to describe acoustic dispensing for the preparation of recombinant and synthetic peptides and we have previously reported the improved accuracy compared to conventional tip-based methods1.
This article describes the integration of the preparation of peptide serial and direct dilutions by non-contact acoustic transfer onto a fully automated plate handling robotics system. A number of methods encompassing acoustic transfer of samples have been described previously11. We utilize a two-step method to prepare intermediate stock concentrations and to serially dilute peptide analogues for the generation of the full dose-response curve. The prepared peptides are incubated with cells expressing the target mouse GLP-1R, and we use a commercially available homogenous time-resolved fluorescence (HTRF) assay to measure cAMP accumulation within these cells as a readout of peptide agonist activity. The assay is robust and amenable to a high-throughput 384-well format and routinely applied to both assay development and drug screening projects12.

<table border="0" cellpadding="0" cellspacing="0" width="1339">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hanks&#8217; Balanced Salt solution</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8264</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HEPES</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bovine Serum Albumin</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7018</td>
			<td>Prepared as a 0.5 M stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">GLP-1 (7-36) amide</td>
			<td style="width:235px;">Bachem</td>
			<td>H-6795</td>
			<td>Prepared as a 1 mg/ml stock in PBS, referred to as &#39;100X reference control&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Test peptides</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Supplied at various concentrations in DMSO or PBS as appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">100X peptide stock</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Test peptide diluted into assay buffer to 100X final required concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:235px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cedex XS Cell Analyzer</td>
			<td style="width:235px;">Innovatis</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Corning 384 well plates, low volume</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">4514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified 384-Well Polypropylene Microplate</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">P-05525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified Reservoir</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">ER-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 550 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 2.5 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 525 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 25 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">ACell Benchtop Automation&#160;</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;">MC522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cellario Lab Automation Scheduling software for Life Science Robotics</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">MultidropCombi Reagent Dispenser</td>
			<td style="width:235px;">ThermFisher Scientific</td>
			<td style="width:119px;">5840300</td>
			<td>Referred to as &#39;bulk reagent dispenser&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:235px;">Cisbio Bioassays</td>
			<td style="width:119px;">62AM4PEJ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">EnVision Multilabel Reader</td>
			<td style="width:235px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

We routinely use a two-step method to dilute peptides via acoustic transfer. For the first step, an acoustic dispenser aligned with automation is used to create four stock peptide intermediate dilutions across two source plates (Figure 1a, b). For the second step, we use an acoustic dispenser to further dilute stock dilutions from source plates A and B to create an 11-point concentration range for each test peptide (Figure 1c). Each pepti...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

The overall goal of this automated protocol for the serial dilution of peptide samples is to minimize peptide absorption to plasticware enabling accurate potency determination for drug discovery. This method can increase the quality and reproducibility of data and the removal of tip-based pipetting also eliminates sample carryover so that errors are not propagated throughout the entire serial dilution. The main advantage of this technique is that it increases data quality by minimizing peptide absorption to plasticware.This is particularly important for driving a structure-activity relationship or lead identification campaign in drug discovery. After preparing the assay buffer and creating a dispensing program for a bulk dispenser according to the text protocol, immerse dispensing cassette tubing in assay buffer and prime with fluid. Place a 384 well low volume assay plate on a plate carrier.Then press start. Dispense 25 microliters of 100 times peptide stocks into columns one through five of an acoustically qualified 384 well flat-bottomed polypropylene microplate to be designated as source plate A.Dispense 25 microliters of 100 times reference control into wells A23 and A24. Then dispense 10 microliters of assay buffer into columns 11 through 15 and 30 microliters of assay buffer into columns 21 through 22.Next, into columns six through 10 and 16 through 20 of a second acoustically qualified 384 well polypropylene microplate designated source plate B, dispense 10 microliters of assay buffer. Centrifuge source plates A and B at 300 times g for one minute. Next, load the source and assay plates into the plate hotel section of the robotics.Then running the robotics software according to the text protocol, use acoustic fluid dispensers integrated into the system to prepare three sequential one in 100 intermediate dilutions. Start with transferring from columns one to five on source plate A to columns six to 10 on source plate B.After preparing the first one in 100 dilution, allow automation to transfer source plate B to an integrated centrifuge and spin at 300 times g for one minute before returning the plate to the acoustic fluid dispenser. Then after transferring the solution from source plate B to source plate A for the second one in 100 dilution, automation will transfer source plate A to the centrifuge and spin before returning the plate to the fluid dispenser.Finally, after automation transfers the solution from plate A to B for the third one in 100 dilution, plate B is again centrifuged. These peptide dilutions in source plate A and source plate B are then used to prepare full concentration response curves in the assay as described in the text protocol. This step is critical as a comparative tip-based methodology causes a dramatic loss in sample potency due to peptide absorption to plasticware.Rapidly thaw cryopreserved CHO cells expressing the target mouse GLP1 receptor in a 37 degrees water bath and use 20 milliliters of assay buffer to resuspend them. Centrifuge the cell suspension at 200 times g and room temperature for five minutes. Discard the supernatant and use 10 milliliters of assay buffer to resuspend the cell pellet.With Trypan Blue, dilute the cell stock one to one and use an automated cell counter to determine the viable cell density. Then use assay buffer to resuspend the cells at a concentration of 1.6 million cells per milliliter. Using a bulk reagent dispenser, add five microliters of cell suspension to each well of the assay plate containing serially diluted peptides and incubate at room temperature for 30 minutes.To carry out the cyclic AMP detection assay, use the lysis buffer to prepare one in 20 dilutions of each homogenous time-resolved fluorescence or HTRF reagent. Use a bulk reagent dispenser to add five microliters of cryptate reagent to all the wells of the assay plates. Then add five microliters of D2 reagent to columns one through 22 of the plate.Next, add five microliters of D2 reagent into wells E23 through P24. Immediately use a manual pipette to add five microliters of lysis buffer to wells A23 through D24 to be used as nonspecific binding control wells. Centrifuge the assay plates at 200 times g and room temperature for one minute to mix the wells.Then cover the plates and incubate for one hour. After the incubation, use a plate reader to measure the fret signal at an excitation of 320 nanometers and an emission of 620 and 665 nanometers. Analyze the data according to the text protocol.The first step in acoustic transfer for the cyclic AMP detection assay is to create four stock peptide intermediate dilutions across two source plates as shown here. For the second step, the acoustic dispenser further dilutes stock solutions from source plates A and B to create an 11-point concentration range for each test peptide in duplicate for 16 different peptides. Assay buffer backfill is performed to ensure a constant volume per well across the assay plate.Shown here is a heat map for an example plate containing 16 peptides in duplicate run through the cyclic AMP accumulation assay. Low values shown in purple represent high cyclic AMP concentration. These concentration response curves illustrate the reproducibility of duplicate points.Sample one represents a positive control for the assay. Samples two, three, and four were inactive and are represented by the flat lines on the graph. Samples five through 16 represent active test peptides.The method presented here allows for the generation of full concentration response curves across a broad potency range. Once mastered, this technique can be completed in three hours, but this will depend on the number of samples requiring preparation. While attempting this procedure, it is important to remember that peptides are biological samples and should be handled as such.In order to maintain their integrity, repeat freeze-thaw cycles should be avoided. This technique will pave the way for researchers in the field of peptide drug discovery and allow for the accurate potency predictions needed to fuel structure-activity relationships. After watching this video, you should have a good understanding of automated acoustic dispensing for the serial dilution of peptides and this will minimize peptide loss by absorption and increase the accuracy of your measurements.Don't forget that working with biologically active peptides with picomolar potencies can be extremely hazardous and precautions such as personal protective equipment should always be used.

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Research

JoVE Journal

Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...