Name: detection of the ph-dependent activity of escherichia coli chaperone hdeb in vitro and in vivo
Uploaded: 2016-10-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo at JoVE.com

We thank Dr. Claudia Cremers for her helpful advice on chaperone assays. Ken Wan is acknowledged for his technical assistance in HdeB purification. This work was supported by the Howard Hughes Medical Institute (to J.C.A.B.) and the National Institutes of Health grant RO1 GM102829 to J.C.A.B. and U.J. J.-U. D. is supported by a postdoctoral research fellowship provided by the German Research Foundation (DFG).

1. Expression and Purification of Periplasmic HdeB
NOTE: HdeB was expressed in E. coli cells harboring the plasmid pTrc-hdeB10, and purified from the periplasm upon polymyxin lysis.
<ol>
	<li>Prepare an overnight culture of E. coli cells harboring the plasmid pTrc-hdeB 10 in 30 ml LB containing 200 &#181;g/ml ampicillin (LBAmp). Inoculate four 1 L cultures of LBAmp and grow them at 37 &#176;C and 200 rpm until O.D.600nm of...

<ol>
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In order to study the mechanism of activation and chaperone function of HdeB, large quantities of HdeB have to be expressed and purified. A number of expression vector systems are available for the production of high levels of a target protein, including pTrc or pBAD vectors, both of which were used in this study. The promoters are readily accessible for E. coli RNA polymerase and thus allow strongly upregulated expression of HdeB in any E. coli strain. This aspect is especially relevant for in vivo...

A common natural environment in which microbial pathogens experience acid-induced protein unfolding conditions is the mammalian stomach (pH range 1-4), whose acidic pH serves as an effective barrier against food-borne pathogens 1. Protein unfolding and aggregation, which is caused by amino acid side chain protonation, affects biological processes, damages cellular structures and eventually causes cell death 1,2. Since the pH of the bacterial periplasm equilibrates almost instantaneously with the environmental pH due to the free diffusion of protons through the porous outer membrane, periplasmic and inner membrane proteins of Gram-negative bacteria are the most vulnerable cellular components under acid-stress conditions 3. To protect their periplasmic proteome against rapid acid-mediated damage, Gram-negative bacteria utilize the acid-activated periplasmic chaperones HdeA and HdeB. HdeA is a conditionally disordered chaperone 4,5: At neutral pH, HdeA is present as a folded, chaperone-inactive dimer. Upon a pH shift below pH 3, HdeA&#39;s chaperone function is quickly activated 6,7. Activation of HdeA requires profound structural changes, including its dissociation into monomers, and the partial unfolding of the monomers 6-8. Once activated, HdeA binds to proteins that unfold under acidic conditions. It effectively prevents their aggregation both during the incubation at low pH as well as upon pH neutralization. Upon return to pH 7.0, HdeA facilitates the refolding of its client proteins in an ATP-independent manner and converts back into its dimeric, chaperone-inactive conformation 9. Similarly, the homologous chaperone HdeB is also chaperone-inactive at pH 7.0. Unlike HdeA, however, HdeB&#39;s chaperone activity reaches its apparent maximum at pH 4.0, conditions under which HdeB is still largely folded and dimeric 10. Moreover, further lowering the pH causes the inactivation of HdeB. These results suggest that despite their extensive homology, HdeA and HdeB differ in their mode of functional activation allowing them to cover a broad pH range with their protective chaperone function. One other chaperone that has been implicated in the acid resistance of E. coli is the cytoplasmic Hsp31, which appears to stabilize unfolded client proteins until neutral conditions are restored. The precise mode of Hsp31&#39;s action, however, has remained enigmatic 12. Given that other enteropathogenic bacteria such as Salmonella lack the hdeAB operon, it is very likely that other yet unidentified periplasmic chaperones might exist that are involved in acid resistance of these bacteria 11.
The protocols presented here allow to monitor the pH-dependent chaperone activity of HdeB in vitro and in vivo 10 and can be applied to investigate other chaperones such as Hsp31. Alternatively, the complex network of transcription factors that control the expression of hdeAB can potentially be investigated by the in vivo stress assay. To characterize the chaperone function of proteins in vivo, different experimental setups can be applied. One route is to apply protein unfolding stress conditions and phenotypically characterize mutant strains that either overexpress the gene of interest or carry a deletion of the gene. Proteomic studies can be conducted to identify which proteins no longer aggregate under stress conditions when the chaperone is present, or the influence of a chaperone on a specific enzyme can be determined during stress conditions using enzymatic assays 14-16. In this study, we chose to overexpress HdeB in an rpoH deletion strain, which lacks the heat shock sigma factor 32. RpoH controls the expression of all major E. coli chaperones and its deletion is known to increase sensitivity to environmental stress conditions that cause protein unfolding 15. The in vivo chaperone activity of HdeB was determined by monitoring its ability to suppress the pH sensitivity of the &#916;rpoH strain. Altogether, the protocols presented here provide a fast and straightforward approach to characterize the activity of an acid-activated chaperone in vitro as well as in the in vivo context.

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			<td height="63" style="height:63px;width:216px;">NEB10-beta E. coli cells</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">C3019I</td>
			<td style="width:1791px;"></td>
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			<td height="63" style="height:63px;width:216px;">Ampicillin</td>
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			<td height="42" style="height:42px;width:216px;">LB Broth mix, Lennox</td>
			<td style="width:64px;">LAB Express</td>
			<td style="width:119px;">3003</td>
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			<td height="63" style="height:63px;width:216px;">IPTG</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">I2481C50</td>
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			<td height="63" style="height:63px;width:216px;">Sodium chloride</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">S271-10</td>
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			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:64px;">Amresco</td>
			<td style="width:119px;">0826-5kg</td>
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			<td height="63" style="height:63px;width:216px;">EDTA</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
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			<td height="63" style="height:63px;width:216px;">Polymyxin B sulfate&#160;</td>
			<td style="width:64px;">ICN Biomedicals Inc.</td>
			<td style="width:119px;">100565</td>
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			<td height="42" style="height:42px;width:216px;">0.2 UM pore sterile Syringe Filter</td>
			<td style="width:64px;">Corning</td>
			<td style="width:119px;">431218</td>
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			<td height="84" style="height:84px;width:216px;">HiTrap Q HP (CV 5 ml)</td>
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			<td style="width:119px;">17-1153-01</td>
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			<td height="21" style="height:21px;width:216px;">Mini-Protean TGX, 15%</td>
			<td style="width:64px;">Bio-Rad</td>
			<td style="width:119px;">4561046</td>
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			<td height="21" style="height:21px;width:216px;">Malate dehydrogenase (MDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127914001</td>
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			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Monobasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-500</td>
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			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Dibasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP363-1</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">F-4500 fluorescence spectrophotometer</td>
			<td style="width:64px;">Hitachi</td>
			<td style="width:119px;">FL25</td>
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			<td height="21" style="height:21px;width:216px;">Oxaloacetate</td>
			<td style="width:64px;">Sigma</td>
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			<td height="21" style="height:21px;width:216px;">NADH</td>
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			<td height="21" style="height:21px;width:216px;">Sodium phosphate monobasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S9390-2.5KG</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S397-500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactate dehydrogenase (LDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127230001</td>
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			<td height="63" style="height:63px;width:216px;">Beckman Proteome Lab XL-I analytical Ultracentrifuge</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">392764</td>
			<td>https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&#38;_windowLabel=UCM_RENDERER&#38;_urlType=render&#38;wlpUCM_RENDERER_path=%252Fwsr%252Fresearch-and-discovery%252Fproducts-and-services%252Fcentrifugation%252Fproteomelab-xl-a-xl-i%252Findex.htm#2/10//0/25/1/0/asc/2/392764///0/1//0/%2Fwsrportal%2Fwsr%2Fresearch-and-discovery%2Fproducts-and-services%2Fcentrifugation%2Fproteomelab-xl-a-xl-i%2Findex.htm/</td>
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			<td height="63" style="height:63px;width:216px;">Centerpiece, 12 mm, Epon Charcoal-filled</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">306493</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AN-50 Ti Rotor, Analytical, 8-Place</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">363782</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wizard Plus Miniprep Kit</td>
			<td style="width:64px;">Promega</td>
			<td style="width:119px;">A1470</td>
			<td>used for plasmid purification (Protocol 5.1)</td>
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			<td height="63" style="height:63px;width:216px;">L-arabinose</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">A-300-500</td>
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			<td height="63" style="height:63px;width:216px;">Glycine</td>
			<td style="width:64px;">DOT Scientific Inc</td>
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			<td height="63" style="height:63px;width:216px;">Fluorescence Cell cuvette</td>
			<td style="width:64px;">Hellma Analytics</td>
			<td style="width:119px;">119004F-10-40</td>
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			<td height="42" style="height:42px;width:216px;">Oligonucleotides</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;"></td>
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			<td height="63" style="height:63px;width:216px;">Phusion High-Fidelity DNA polymerase</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">dNTP set</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;">10297018</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrochloric Acid</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">A144-212</td>
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			<td height="63" style="height:63px;width:216px;">Sodium Hydroxide</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra 15 mL 3K NMWL</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC900324</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge Avanti J-26XPI</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">393127</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Varian Cary 50 spectrophotometer</td>
			<td style="width:64px;">Agilent Tech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa</td>
			<td style="width:64px;">Spectrum Laboratories</td>
			<td style="width:119px;">132650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra Centrifugal Filter Units 30K</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC803024</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SDS</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">bp166-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Veriti 96-Well Thermal Cycler</td>
			<td style="width:64px;">Thermo Fisher</td>
			<td style="width:119px;">4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of the ph-dependent activity of escherichia coli chaperone hdeb in vitro and in vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

HdeA and HdeB are homologous E. coli proteins, known to protect periplasmic proteins against acid stress conditions 10. Our work revealed that similar to HdeA, HdeB also functions as an acid activated molecular chaperone. However, in contrast to HdeA, HdeB functions at a pH that is still potentially bactericidal, but significantly higher than the pH optimum of HdeA 6,9,10,22. To investigate the pH optimum of HdeB&#39;s chaperone activity in vitro, n...

Watch this Scientific Journal Video about Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo at JoVE.com

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

The overall goal of these in vivo and in vitro chaperone assays is to characterize the pH-dependent activity of the Escherichia coli chaperone HdeB under acidic pH conditions. Acid activated chaperones have only one job to do, they need to protect proteins against acid induced protein unfolding and degradation. This function is particularly important for enteric bacteria, which have to survive the acidic environment of the stomach if they want to successfully colonize the gut.The methods that we are presenting here allow you to identify and characterize acid activated chaperones, and define the pH conditions that lead to their activation. After watching this video, you should have a good understanding of how to characterize the chaperones, such as HdeB in vitro and in vivo, which helps you to decipher the mechanism of its activation. These methods have been successfully applied for other acid protective chaperones, such as HDA, and can be further modified to work with other chaperones, and/or stress conditions.The influence of purified HdeB on the aggregation of thermally unfolding porcine mitochondrial malate dehydrogenase at different pH values is monitored. Dialyzed MDH at four degrees Celsius over night against four liters of buffer C.Then concentrate the protein to approximately 100 micromolar using centrifugal filter units with a molecular weight cutoff of 30 kilodaltons. To remove aggregates, centrifuge the protein for 20 minutes at 20, 000 times G at four degrees Celsius.Determine MDH concentration by absorbance at 280 nanomolar. Then prepare 50 micro liter aliquots of MDH and flash-freeze the aliquots for storage. Place a one milliliters quartz cuvette into a florescent spectrophotometer equipped with temperature controlled sample holders and stirrers.Set the excitation and admission wavelength to 350 nanometers. Add the appropriate volumes of pre-warmed buffer D at the desired pH values to the cuvette. The total volume is 1, 000 microliters.Set the temperature in the cuvette holder to 43 degrees Celsius. Add 12.5 micromolar HdeB to the buffer, followed by the addition of 0.5 micromolar MDH. Begin monitoring light scattering.Incubate the reaction for 360 seconds to allow sufficient unfolding of MDH. Raise the pH to seven by adding a 0.16 to 0.34 volume of two molar un-buffered dipotassium phosphate, and continue recording light scattering for another 440 seconds. Analyze the data as described in the text protocol.Transform the plasmid expressing HdeB or the MD vector control, PBAD 18, into strain BB7224 Delta RPOH using chemically competent cells. This strain is temperature sensitive and has to be cultivated at 30 degrees Celsius. After 45 seconds of heat shock at 42 degrees Celsius and prior to plating, incubate the cells at 30 degrees Celsius and 200 RPM.Perform single colony streak outs of positive clones, and incubate over night at 30 degrees Celsius. Prepare an over night culture in 50 milliliters of LB with Ampicillin, and cultivate the cells at 200 RPM and 30 degrees Celsius. Dilute over night cultures 40 fold into 25 milliliters of LB with Ampicillin.Grow the bacteria in the presence of 0.5%arabinose at 30 degrees Celsius and 200 RPM to an optical density at 600 nanometers, or OD 600 equal to 1.0, to induce HDEB protein expression. For the pH shift experiments, use LB with Ampicillin and arabinose to dilute the cells to an OD 600 of 0.5. Adjust to the respective pH values by adding appropriate volumes of five molar hydrochloric acid.After the indicated time points, neutralize the cultures by addition of the appropriate volumes of five molar sodium hydroxide. Monitor the growth of the neutralized cultures and liquid culture for 12 hours at 30 degrees Celsius using OD measurements. To investigate the pH optimum of HdeB's chaperone activity in vitro, native MDH was diluted into pre-warmed buffer of the indicated pH in the presence, or absence, or HdeB.Following 360 seconds of incubation, aggregation of MDH at 43 degrees Celsius was triggered through neutralization. In the presence of HdeB, the light scattering signal is significantly decreased upon neutralization from pH four, indicating that HdeB prevents the aggregation of MDH. Aggregation measurements are very sensitive to changes in temperature or buffer content.To eliminate false positive results, the influence of chaperone storage buffer on client aggregation was investigated. The HdeB storage buffer had no effect on MDH aggregation. To investigate the effects of HdeB in vivo, pH dependent survival assays were conducted using the temperature sensitive RPOH deletion strain.This strain lacks most chaperones, and is therefore more susceptible to elevated temperature, low pH, or oxidated stress. Over expression of the HdeB under neutral pH conditions showed no effect on the growth rate of the strain, which grew comparably well to the control strain that harbors the empty vector, PBAD 18. In contrast, clear differences were found in their ability to resume growth upon pH three or pH four treatments, with the HdeB over expressing strain showing reproducibly improved recovery from low pH treatment as compared to the control strain.Once mastered, the purification can be done in three days, the in vitro scattering assay in one day, and the in vivo survival assays in two days if it is performed properly. When you're doing these experiments, make sure that you consider your experimental design carefully, both in regards to the type of client proteins, their concentrations, and the buffer conditions that you use. The main advantage of this technique is that it helps to gain a more detailed understanding about the mechanism of chaperone activation and its effects in an in vivo context.And don't forget that studying the pH optimum of an acid protective chaperone can quite challenging. This is because the aggregation behavior of the client protein that you use will likely also change with the pH. So therefore, make sure to choose buffer systems that are suitable for both the chaperone and the client protein.Following this procedure, other methods like spin down assays by SCS page, can be performed in auto quantified chaperone mediated suppression of client aggregation.

detection-ph-dependent-activity-escherichia-coli-chaperone-hdeb-vitro

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Defining Hsp33&#039;s Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.