Name: application of biolayer interferometry (bli) for studying protein-protein interactions in transcription
Uploaded: 2019-07-26T13:00:00
Description: Watch this Scientific Journal Video about Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription at JoVE.com

This work was supported by National Institutes of Health (Grants # AI122034 and AI140167) and New Jersey Health Foundation (Grant # PC 20-18).

1. Preparation of proteins
<ol>
	<li>Use a dialysis bag (with an appropriate cut-off size) to dialyze each protein to be used for BLI assays (including both the His-tagged ligand and the analyte) against 1,000 volumes of the BLI buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 10 mM MgCl2, 0.1 mM DTT, pH 8.0, pre-chilled to 4 &#730;C) at 4 &#730;C for 4 h. 
	NOTE: BLI assays require the ligand to be present at concentrations that saturate the binding sites on the biosensor and the analyte to be highly ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+bacterial+transcription+factors:+how+proteins+take+on+new+tasks,+but+do+not+always+stop+doing+the+old+ones.">Evolution of bacterial transcription factors: how proteins take on new tasks, but do not always stop doing the old ones.</a> Trends in Microbiology. 23 (8), 463-467 (2015).</li><li>Taylor, H. R., Burton, M. J., Haddad, D., West, S., Wright, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trachoma.">Trachoma.</a> Lancet. 384 (9960), 2142-2152 (2014).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+incidence+and+prevalence+of+selected+curable+sexually+transmitted+infections:+2008.">Global incidence and prevalence of selected curable sexually transmitted infections: 2008.</a> Sexual and Reproductive Health Matters. 20, (2012).</li><li>Schmidt, S. M., Muller, C. E., Mahner, B., Wiersbitzky, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+rate+of+persistence+and+respiratory+tract+symptoms+of+Chlamydia+pneumoniae+infection+in+1211+kindergarten+and+school+age+children.">Prevalence, rate of persistence and respiratory tract symptoms of Chlamydia pneumoniae infection in 1211 kindergarten and school age children.</a> The Pediatric Infectious Disease Journal. 21 (8), 758-762 (2002).</li><li>De Puysseleyr, 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=Evaluation+of+the+presence+and+zoonotic+transmission+of+Chlamydia+suis+in+a+pig+slaughterhouse.">Evaluation of the presence and zoonotic transmission of Chlamydia suis in a pig slaughterhouse.</a> BMC Infectious Diseases. 14 (560), (2014).</li><li>Hulin, V., 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=Host+preference+and+zoonotic+potential+of+Chlamydia+psittaci+and+C.+gallinacea+in+poultry.">Host preference and zoonotic potential of Chlamydia psittaci and C. gallinacea in poultry.</a> Pathogens and Disease. 73 (1), 1-11 (2015).</li><li>Belland, R., Ojcius, D. M., Byrne, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia.">Chlamydia.</a> Nature Review of Microbiol. 2 (7), 530-531 (2004).</li><li>Belland, R. 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=Genomic+transcriptional+profiling+of+the+developmental+cycle+of+Chlamydia+trachomatis.">Genomic transcriptional profiling of the developmental cycle of Chlamydia trachomatis.</a> Proceedings of the National Academy of Sciences of the USA. 100 (14), 8478-8483 (2003).</li><li>Nicholson, T. L., Olinger, L., Chong, K., Schoolnik, G., Stephens, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+stage-specific+gene+regulation+during+the+developmental+cycle+of+Chlamydia+trachomatis.">Global stage-specific gene regulation during the developmental cycle of Chlamydia trachomatis.</a> Journal of Bacteriology. 185 (10), 3179-3189 (2003).</li><li>Tan, M., Tan, M., Bavoil , P. 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> Intracellular pathogens I: Chlamydiales. , 149-169 (2012).</li><li>Domman, D., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+the+Footsteps+of+Chlamydial+Gene+Regulation.">Following the Footsteps of Chlamydial Gene Regulation.</a> Molecular Biology and Evolution. 32 (12), 3035-3046 (2015).</li><li>Bao, X., Nickels, B. E., Fan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis+protein+GrgA+activates+transcription+by+contacting+the+nonconserved+region+of+&#963;66.">Chlamydia trachomatis protein GrgA activates transcription by contacting the nonconserved region of &#963;66.</a> Proceedings of the National Academy of Sciences of the USA. 109 (42), 16870-16875 (2012).</li><li>Desai, 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=Role+for+GrgA+in+regulation+of+&#963;28-dependent+transcription+in+the+obligate+intracellular+bacterial+pathogen+Chlamydia+trachomatis.">Role for GrgA in regulation of &#963;28-dependent transcription in the obligate intracellular bacterial pathogen Chlamydia trachomatis.</a> Journal of Bacteriology. 200 (20), (2018).</li><li>Joy, C., 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=Label-Free+Detection+of+Biomolecular+Interactions+Using+BioLayer+Interferometry+for+Kinetic+Characterization.">Label-Free Detection of Biomolecular Interactions Using BioLayer Interferometry for Kinetic Characterization.</a> Combinatorial Chemistry &amp; High Throughput Screening. 12 (8), 791-800 (2009).</li><li>Abdiche, Y., Malashock, D., Pinkerton, A., Pons, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+kinetics+and+affinities+of+protein+interactions+using+a+parallel+real-time+label-free+biosensor,+the+Octet.">Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet.</a> Analytical Biochemistry. 377 (2), 209-217 (2008).</li><li>Szabo, A., Stolz, L., Granzow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+and+its+use+in+biomolecular+interaction+analysis+(BIA).">Surface plasmon resonance and its use in biomolecular interaction analysis (BIA).</a> Current Opinion in Structural Biology. 5 (5), 699-705 (1995).</li><li>Nelson, R. W., Krone, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+surface+plasmon+resonance+biomolecular+interaction+analysis+mass+spectrometry+(BIA/MS).">Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS).</a> Journal of Molecular Recognition. 12 (2), 77-93 (1999).</li><li>Yang, D., Singh, A., Wu, H., Kroe-Barrett, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+biosensor+platforms+in+the+evaluation+of+high+affinity+antibody-antigen+binding+kinetics.">Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics.</a> Analytical Biochemistry. 508, 78-96 (2016).</li><li>Visentin, 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=Overcoming+non-specific+binding+to+measure+the+active+concentration+and+kinetics+of+serum+anti-HLA+antibodies+by+surface+plasmon+resonance.">Overcoming non-specific binding to measure the active concentration and kinetics of serum anti-HLA antibodies by surface plasmon resonance.</a> Biosensors and Bioelectronics. 117, 191-200 (2018).</li><li>Baba, A., Taranekar, P., Ponnapati, R. R., Knoll, W., Advincula, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+surface+plasmon+resonance+and+waveguide-enhanced+glucose+biosensing+with+N-alkylaminated+polypyrrole/glucose+oxidase+multilayers.">Electrochemical surface plasmon resonance and waveguide-enhanced glucose biosensing with N-alkylaminated polypyrrole/glucose oxidase multilayers.</a> ACS Applied Materials &amp; Interfaces. 2 (8), 2347-2354 (2010).</li><li>Del Vecchio, K., Stahelin, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Surface+Plasmon+Resonance+to+Quantitatively+Assess+Lipid-Protein+Interactions.">Using Surface Plasmon Resonance to Quantitatively Assess Lipid-Protein Interactions.</a> Methods in Molecular Biology (Clifton, N.J.). 1376, 141-153 (2016).</li><li>Wang, D. S., Fan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Surface+Plasmon+Resonance+Sensors:+From+Principles+to+Point-of-Care+Applications.">Microfluidic Surface Plasmon Resonance Sensors: From Principles to Point-of-Care Applications.</a> Sensors (Basel, Switzerland). 16 (8), 1175 (2016).</li><li>Shah, N. B., Duncan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-layer+interferometry+for+measuring+kinetics+of+protein-protein+interactions+and+allosteric+ligand+effects.">Bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects.</a> Journal of visualized experiments : JoVE. (84), e51383 (2014).</li><li>Wartchow, C. 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=Biosensor-based+small+molecule+fragment+screening+with+biolayer+interferometry.">Biosensor-based small molecule fragment screening with biolayer interferometry.</a> Journal of Computer-Aided Molecular Design. 25 (7), 669-676 (2011).</li></ol>

Protein-protein interactions are crucial for the regulation of transcription and other biological processes. They are most commonly studied through pulldown assays. Although pulldown assays are relatively easy to perform, they are poorly quantitative and may fail to detect weak but biologically meaningful interactions. In comparison, by detecting real-time association and dissociation between a ligand and an analyte, BLI provides association and dissociation rate constants, as well as, overall affinity.
<p class="jov...

Transcription, which produces RNA molecules using DNA as template, is the very first step of gene expression. Bacterial RNA synthesis begins following the binding of the RNA polymerase (RNAP) holoenzyme to a target promoter1,2. The RNAP holoenzyme (RNAPholo) is comprised of a multi-subunit catalytic core (RNAPcore) and a &#963; factor, which is required for recognizing the promoter sequence. Transcription activators and repressors, collectively termed TFs, regulate the gene expression through the binding components of the RNAPcore, &#963; factors, and/or DNA. Depending on the organism, a significant portion of its genome may be devoted to TFs that regulate transcription in response to physiological needs and environmental cues3.
Chlamydia is an obligate intracellular bacterium responsible for a variety of diseases in humans and animals4,5,6,7,8. For example, Chlamydia trachomatis is arguably the number one sexually transmitted pathogen in humans worldwide, and a leading cause of blindness in some underdeveloped countries4,5. Chlamydia has a unique developmental cycle characterized by two alternating cellular forms termed the elementary body (EB) and reticulate body (RB)9. Whereas, EBs are capable of survival in an extracellular environment, they are incapable of proliferation. EBs enter host cells through endocytosis and differentiate into larger RBs in a vacuole in the host cytoplasm within hours post-inoculation. No longer infectious, RBs proliferate through binary fission. Around 20 h, they start to differentiate back to the EBs, which exit the host cells around 30-70 h.
Progression of the chlamydial developmental cycle is regulated by transcription. Whereas a supermajority of the nearly 1,000 chlamydial genes are expressed during the midcycle during which RBs are actively replicating, only a small number of genes are transcribed immediately after the entry of EBs into the host cells to initiate the conversion of EBs into RBs, and another small set of genes are transcribed or increasingly transcribed to enable the differentiation of RBs into EBs10,11.
The chlamydial genome encodes three &#963; factors, namely &#963;66, &#963;28 and &#963;54. &#963;66, which is equivalent to the housekeeping &#963;70 of E. coli and other bacteria, is responsible for recognizing promoters of early and mid-cycle genes as well as some late genes, whereas &#963;28 and &#963;54 are required for the transcription of certain late genes. Several genes are known to carry both a &#963;66-dependent promoter and a &#963;28-dependent promoter12.
Despite a complicated developmental cycle, only a small number of TFs have been found in chlamydiae13. GrgA (previously annotated as a hypothetical protein CT504 in C. trachomatis serovar D and CTL0766 in C. trachomatis L2) is a Chlamydia-specific TF initially recognized as an activator of &#963;66-dependent genes14. Affinity pulldown assays have demonstrated that GrgA activates their transcription by binding both &#963;66 and DNA. Interestingly, it was later found with that GrgA also co-precipitates with &#963;28, and activates transcription from &#963;28-dependent promoters in vitro15. To investigate whether GrgA has similar or different affinities for &#963;66 and &#963;28, we resorted to using BLI. BLI assays have shown that GrgA interacts with &#963;66 at a 30-fold higher affinity than with &#963;28, suggesting that GrgA may play differential roles in &#963;66-dependent transcription and &#963;28-dependent transcription15.
BLI detects the interference pattern of white light that reflects from a layer of immobilized protein on the tip of a biosensor and compares it to that of an internal reference layer16. Through the analysis of these two interference patterns, BLI can provide valuable and real-time information about the amount of protein bound to the tip of the biosensor. The protein that is immobilized to the tip of the biosensor is referred to as the ligand, and is generally immobilized with the help of a common antibody or epitope tag (e.g., a poly-His- or biotin-tag) that has an affinity for an associated particle (such as NTA or Streptavidin) on the tip of the biosensor. The binding of a secondary protein, referred to as the analyte, with the ligand at the tip of the biosensor creates changes in the opacity of the biosensor and therefore results in changes in interference patterns. When repeated over different concentrations of the analyte, BLI can provide not only qualitative but also quantitative information about the affinity between the ligand and analyte16.
To the best of our knowledge, we were the first to employ BLI to characterize protein-protein interactions in transcription15. In this publication, we demonstrate that a GrgA fragment, which was previously shown to be required for &#963;28-binding, indeed mediates the binding. This manuscript focuses on steps of the BLI assays, and generation of BLI graphs and parameters of binding kinetics. Methods for the production (and purification) of ligands and analytes are not covered here.

<table>
	<tbody>
		<tr>
			<td>BLItz machine</td>
			<td>ForteBio</td>
			<td>45-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>MilliporeSigma</td>
			<td>D9652</td>
			<td></td>
		</tr>
		<tr>
			<td>Dip and Read Ni-NTA biosensor tray</td>
			<td>ForteBio</td>
			<td>18-5101</td>
			<td>Ready-to-use Ni-NTA biosensors for poly-His-tagged Proteins</td>
		</tr>
		<tr>
			<td>Drop holder</td>
			<td>ForteBio</td>
			<td>45-5004</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes (0.2 mL)</td>
			<td>Thomas Scientific</td>
			<td>CLS6571</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (black)</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-391-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Thermo Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0861</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>MilliporeSigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>MilliporeSigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>MilliporeSigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>GoldBio</td>
			<td>T095100</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of biolayer interferometry (bli) for studying protein-protein interactions in transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Through BLI assays, we previously established that binding of GrgA to &#963;28 is dependent on a 28 amino acid middle region (residues 138-165) of GrgA15. Accordingly, compared with N-terminally His-tagged full length GrgA (NH-GrgA), a GrgA deletion construct lacking this region (NH-GrgA&#916;138-165) had a decreased association rate and an increased dissociation rate, leading to a 3 million-fold loss of overall affinity (Table 1). Here, we demonstrate that this middle ...

Watch this Scientific Journal Video about Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription at JoVE.com

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

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