The authors thank Ms.Alisha Knudson for the editorial help. They would also like to acknowledge the financial support from the NIH/NCI (3R01CA115483, to K.S.L.), the DoD PRMRP Award (W81XWH-13-1-0490, to K.S.L.), the NIH/NCI (1R01CA199668, to Y.L.), and the NIH/NICHD (1R01HD086195, to Y.L.).

Ethics statement: Female athymic nude mice (Nu/Nu strain), 6-8 weeks old, were purchased and then kept under pathogen-free conditions according to AAALAC guidelines and were allowed to acclimatize for at least 4 days prior to any experiments. All animal experiments were performed in compliance with institutional guidelines and according to protocol No. 07-13119 and No. 09-15584, approved by the Animal Use and Care Administrative Advisory Committee at the University of California, Davis.
1. Synthesis of TD PEG5K-Cys4-Ebes8-CA8
<ol>
	<li>In a round-bottom flask, dissolve MeO-PEG5K-NH2 (2 g, 0.4 mmol) in 10-20 ml of anhydrous dimethylformamide (DMF) and chill on ice.</li>
	<li>In a glass beaker, dissolve 3 equiv. of 1-hydroxy-6-chloro-benzotriazole (HOBt), 3 equiv. of N,N&#39;-diisopropylcarbodiimide (DIC), and 3 equiv. of (Fmoc)2Lys-OH in anhydrous DMF (10-15 ml). Stir for 15-20 min on a magnetic stir plate.</li>
	<li>Add the mixture to the reaction flask containing the MeO-PEG5K-NH2. Remove the ice bath and stir the reaction mixture overnight at room temperature.</li>
	<li>Confirm the completion of the reaction with thin layer chromatography (TLC)16 and Kaiser&#39;s test17 (a yellow color indicates the absence of free NH2). Precipitate the polymeric product 1 (MeO-PEG5K-Lys(NH-Fmoc)2) by adding approximately 200 ml of ice-cold ether to the reaction flask. Separate the precipitated polymer via centrifugation (6 min at 6,000 x g and 4 &#176;C).
	<ol>
		<li>To perform TLC, spot the samples on silica gel-coated TLC plates. Use dichloromethane/methanol (9:1) as the mobile phase. Observe spots under a UV lamp after developing the TLC plates. One can also use ninhydrin staining reagent to visualize amine spots on a hot plate.</li>
		<li>For Kaiser&#39;s test, place a little bit of the sample in glass tube containing Kaiser&#39;s reagent. Heat it at 100 &#176;C for 5 min and look for the color change (if the color of the solution remains yellow, then the reaction is complete).</li>
	</ol>
	</li>
	<li>Re-dissolve the product in anhydrous DMF (10-20 ml) and repeat the precipitation and centrifugation (see step 1.4).</li>
	<li>Repeat step 1.5, and then wash the polymer precipitate three times with ice-cold ether.</li>
	<li>Transfer the polymer precipitate 1 to a clean reaction flask and connect the flask to a high vacuum source to remove the residual ether.</li>
	<li>Prepare and add approximately 20-30 ml of 20% (v/v) 4-methylpiperidine in DMF to polymer intermediate 1. Stir until complete dissolution. Run the reaction for 3 hr.</li>
	<li>Perform TLC16 and Kaiser&#39;s test (a blue color confirms the presence of free NH2)17 (see step 1.4) to confirm the completion of the reaction. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6).</li>
	<li>Dry the polymeric product 2 (MeO-PEG5K-Lys(NH2)2) under a vacuum.</li>
	<li>Carry out one more round of (Fmoc)2Lys-OH-coupling onto intermediate 2 (steps 1.1-1.7) to generate product 3 (MeO-PEG5K-Lys(Lys(NH-Fmoc)2)2). De-protect (steps 1.8-1.10) the Fmoc groups (4, MeO-PEG5K-Lys(Lys(NH2)2)2) and couple (steps 1.1-1.7) the (Fmoc)Lys(Boc)-OH to generate a third-generation dendritic polylysine (5, MeO-PEG5k-Lys-Lys2-((Fmoc)Lys(Boc))4) terminated with four Boc and Fmoc groups on one end of the PEG chain.</li>
	<li>Transfer the resulting polymer intermediate 5 to a reaction flask. In a separate reaction flask, prepare 1:1 (v/v) trifluoroacetic acid (TFA) in dichloromethane (DCM). Add 15-20 ml of a 1:1 TFA/DCM (v/v) mixture to the polymer intermediate 5. Stir the mixture until the polymer is completely dissolved. Stir for an additional 3 hr.</li>
	<li>Perform TLC16 and Kaiser&#39;s test (a blue color confirms the presence of free NH2)17 (see step 1.4) to confirm the completion of the reaction. If the reaction is complete, evaporate the polymer-in-TFA/DCM mixture with air until a viscous solution is obtained. Proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6). Dry the polymeric product 6 (MeO-PEG5k-Lys-Lys2-((Fmoc)Lys(NH2))4) under a vacuum.</li>
	<li>Transfer polymer intermediate 6 into a reaction flask. Use approximately 40 ml of anhydrous DMF containing 8 equiv. of N,N-diisopropylethylamine (DIEA) to dissolve the polymer intermediate 6. In a glass beaker, dissolve 12 equiv. of HOBt, 12 equiv. of DIC, and 12 equiv. of (Fmoc)Cys(Trt)-OH in 20-25 ml of anhydrous DMF. Shake for 10-15 min, and then add the reaction mixture to the reaction flask containing 6. Run the reaction overnight.</li>
	<li>Confirm the completion of reaction with TLC16 and Kaiser&#39;s test (see step 1.4; a yellow color indicates the absence of free NH2)17. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (step 1.4-1.6), to isolate product 7 (MeO-PEG5K-Lys-Lys2-((Fmoc)Lys-((Fmoc)Cys(Trt)))4.</li>
	<li>Perform Fmoc de-protection on 7, as outlined in step 1.8, to obtain product 8 (PEG5K-Lys-Lys2-(Lys(NH2)-(Cys(NH2)(Trt)))4). Couple (Fmoc)-PEG2-Suc-OH (&#34;Ebes&#34; linker, 24 equiv.) on a polymer intermediate using the procedure outlined above for HOBt/DIC-mediated coupling to obtain intermediate 9 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4(Ebes(NH-Fmoc))8).</li>
	<li>Perform one more round of Fmoc de-protection (steps 1.6-1.8) to get intermediate 10 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4(Ebes(NH2))8).</li>
	<li>Transfer the polymer intermediate 10 into a reaction flask and add anhydrous DMF (approximately 30-40 ml) to dissolve it. In another reaction flask, dissolve 24 equiv. of CAOSu (prepared according to the previously published procedure) in anhydrous DMF (20-30 ml)18. Add 48 equiv. of N,N-diisopropylethylamine and let it stir for 10-15 min. Transfer the contents into the reaction flask containing 10 and let the reaction run overnight.</li>
	<li>Confirm the completion of the reaction with TLC16 and Kaiser&#39;s test (see step 1.4; a yellow color indicates the absence of free NH2)17. If the reaction is complete, proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6) to isolate product 11 (MeO-PEG5K-Lys-Lys2-Lys4-(Cys(Trt))4-Ebes8-CA8). Dialyze it in de-ionized water and lyophilize the sample to yield a white powder.</li>
	<li>Place the polymer intermediate 11 into a reaction flask. Prepare and add 20 ml of the TFA/1,2-ethanedithiol (EDT)/triethylsilane (TIS)/H2O (94/2.5/1/2.5, v/v) mixture into the polymer solution. Stir the mixture until complete dissolution. Run the reaction for 4 hr. Confirm the completion of the reaction by TLC16.</li>
	<li>Under the fume hood, blow air into the polymer-TFA/EDT/TIS/H2O mixture until the solution becomes viscous. Proceed to the ether precipitation, as mentioned for product 1 (steps 1.4-1.6), to isolate the final product, 12 (PEG5K-Cys4-Ebes8-CA8). Dissolve it in acetonitrile and lyophilize it to yield a white powder.</li>
</ol>
2. Preparation of PTX-loaded Micelles
<ol>
	<li>Prepare a PTX-loaded PEG5K-Cys4-Ebes8-CA8 micelle using the standard evaporation method.
	<ol>
		<li>Dissolve 20 mg of TD with a different amount of PTX (1-9 mg) in 1 ml of chloroform (CHCl3). Remove the solvent using a rotary evaporator to obtain a homogeneous, dry polymer film. Reconstitute the film with 1 ml of phosphate-buffered saline (PBS) by vortex, followed by sonication for 30 min at 40 kHz, if necessary, to allow the formation of drug-loaded micelles.</li>
		<li>Add 6 &#181;l of 3% (w/w) H2O2 (1 equiv. to free the thiol groups) to oxidize the thiol groups on the TD. Use the micelle solution for further characterization once the level of free thiol groups remains at constant low values, as indicated by Ellman&#39;s test19.</li>
		<li>Filter the solution with a 0.22-&#181;m filter to sterilize the sample. Analyze the amount of drug loaded in the micelles on a HPLC20 system after releasing the drugs from the micelles by adding 9 times acetonitrile and performing 10 min of sonication. Use a C18 column for HPLC with acetonitrile/water as the mobile phase.</li>
		<li>Calculate the drug loading according to the calibration curve between the HPLC area values and the concentrations of the drug standard11. 
		NOTE: The loading efficiency is defined as the ratio of drug loaded into the micelles to the initial drug content.</li>
	</ol>
	</li>
</ol>
3. Characterizations of Micelles
<ol>
	<li>Measure the size and size distribution of the micelles with a dynamic light scattering (DLS) instrument29. Perform the measurements at room temperature and keep the micelle concentration at 1 mg/ml. 
	NOTE: To perform particle size analysis, use PBS as blank, and then record the particle size for actual samples. Take the readings in triplicate for samples, and then average the readings.</li>
	<li>Use fluorescence spectra to measure the critical micellar concentration (CMC) of PEG5K-Cys4-Ebes8-CA8 before and after cross-linking with pyrene as a hydrophobic fluorescent probe, as described previously13,21. 
	NOTE: Typically, the micelle concentration ranges from 5&#215;10-7 to 5&#215;10-4 M.</li>
</ol>
4. Stability of Micelles in SDS with or without Reducing Agents
<ol>
	<li>Prepare stock solutions of sodium dodecyl sulfate (SDS) solution (7.5 mg/ml) and disulfide cross-linked micelles (1.5 mg/ml) in PBS. Next, using the stock solutions, make a solution mixture in which the final SDS concentration is at 2.5 mg/ml and the micelle concentration is at 1.0 mg/ml.</li>
	<li>Measure the size and size distribution (as mentioned in step 3.1) of the micelle solutions at predetermined time intervals with or without the presence of 10 mM glutathione (GSH).</li>
</ol>
5. Hemolysis Assay
<ol>
	<li>Evaluate the hemolytic potential of PTX-loaded, non-cross-linked micelles (PTX-NCMs) prepared according to previously published procedure11 and PTX-loaded cross-linked micelles (PTX-DCMs) using fresh citrated blood from nude mice collected from the tail veil.
	<ol>
		<li>Collect red blood cells by centrifugation of blood sample (1.0 ml) at 1,000 x g for 10 min, wash them three times with PBS, and then re-suspend the cell pellets with PBS to a final concentration of 2%.</li>
	</ol>
	</li>
	<li>Mix 200 &#181;l of erythrocyte suspension with different concentrations (0.2 and 1.0 mg/ml) of PTX-NCMs and PTX-DCMs, respectively, and incubate for 4 hr at 37 &#176;C in an incubator shaker.</li>
	<li>Centrifuge the micelle-erythrocyte mixtures at 3,000 x g for 5 min. Transfer 100 &#181;l of the supernatant of all samples to a 96-well plate. Measure the absorbance of free hemoglobin in the supernatant at 540 nm using a micro-plate reader. 
	NOTE: RBCs incubated with Triton-100 (2%) and PBS are to be used as the positive and negative controls, respectively. The percent hemolysis of the RBCs is calculated with the following formula: RBC hemolysis = (ODsample - ODnegative control)/(ODpositive control - ODnegative control) &#215; 100%.</li>
</ol>

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Y. Li and K.S.L. are the inventors of a pending patent on reversibly crosslinked micelle systems (US patent application 61/485,774). K.S.L. is the founding scientist of LamnoTherapeutics Inc., which plans to develop the nanotherapeutics described in the manuscript. The remaining authors declare no competing financial interests.

Several nanoparticles have been investigated for their potential use in drug delivery. Liposomal doxorubicin and paclitaxel (PTX)-loaded human serum albumin nano-aggregates are among the nanotherapeutics approved by the FDA for cancer treatment. However, although clinically effective, both of these nanotherapeutics are relatively &#34;large&#34; in size, and they tend to accumulate in the liver and lungs. Polymeric micelles with relatively smaller particle sizes and higher drug loading capacities are emerging nanocarrier...

Nanotechnology is a fast-emerging field that has benefited a number of biomedical areas1. Nanoparticles provide opportunities for designing and tuning properties that are not feasible with other types of conventional therapeutics. Nano-carriers enhance the stability of drugs against biodegradation, prolong drug circulation time, overcome drug solubility issues, and can be fine-tuned for targeted drug delivery and for co-delivering imaging agents1,2. Nanoparticle-based delivery systems hold promise in cancer imaging and treatment. Tumor vasculatures are leaky to macromolecules and can lead to preferential accumulation of circulating nanoparticles at tumor sites via the enhanced permeability and retention (EPR) effect3. Among the several nano-carriers (e.g., liposomes, hydrogels, and polymeric micelles) that are being actively pursued as carriers for anti-cancer drugs, polymeric micelles have gained wide popularity over the last decade4,5.
Polymeric micelles are a thermodynamic system that, on intravenous administration, can potentially be diluted below the critical micelle concentration (CMC), leading to their dissociation into unimers. Cross-linking strategies have been employed to minimize micellar dissociation into unimers. However, excessively stabilized micelles may prevent the drug from releasing at the target sites, thereby reducing the overall therapeutic efficacy. Several chemical approaches have been explored to make the cross-linking degradable in response to redox or to external stimuli, such as reducible disulfide bonds6,7 and pH-cleavable8 or hydrolysable ester bonds9,10.
We have previously reported the design and synthesis of micellar nanoparticles consisting of dendritic cholic acid (CA) blocks and linear polyethylene glycol (PEG) copolymers, referred to as telodendrimers (TD)11-15. These TDs are represented as PEGnK-CAy (where n = molecular weight in kilodaltons (K), y = number of cholic acid (CA) units). They are characterized by their small size, long shelf life, and high efficiency in encapsulating drugs such as paclitaxel (PTX) and doxorubicin (DOX) in the hydrophobic core. The building blocks of TD, such as PEG, lysine, and CA, are biocompatible, and the presence of a PEG corona can impart a &#34;stealth&#34; nanoparticle character, preventing non-specific uptake of micellar nanoparticles by the reticuloendothelial systems.
Thiolated linear-dendritic polymers can easily be generated by introducing cysteines into the dendritic oligo-lysine backbone of our standard TDs. This article presents a facile protocol for the production of a reversibly cross-linked micellar drug delivery system by introducing disulfide cross-links into the hydrophobic core of TDs (Figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="795">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">MeO-PEG5K-NH2</td>
			<td style="width:105px;">Rapp Polymere</td>
			<td style="width:119px;">125000-2</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Lys(Fmoc)-OH</td>
			<td style="width:105px;">Aaptec</td>
			<td style="width:119px;">AFK107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Lys(Boc)-OH</td>
			<td style="width:105px;">Anaspec</td>
			<td style="width:119px;">AS-20132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Cys(Trt)-OH</td>
			<td>Aapptec</td>
			<td>AAC105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethylformamide</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">BP1160-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl ether</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">E134-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Diisopropylethylamine</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">D125806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">T6508</td>
			<td>Corrosive, handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-methyl piperidine</td>
			<td style="width:105px;">Alfa-Aesar</td>
			<td style="width:119px;">L-02709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ebes linker</td>
			<td style="width:105px;">Anaspec</td>
			<td>AS-61924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholic acid</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">C1129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,2-Ethanedithiol</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">02390</td>
			<td>Handle inside fume hood. Bleach gloves after usage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">233781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform (anhydrous)</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">288306</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide solution 30%</td>
			<td style="width:105px;">Aaron Industries</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HoBt-Cl</td>
			<td style="width:105px;">Aaptec</td>
			<td style="width:119px;">CXZ096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DIC</td>
			<td style="width:105px;">Sigma Aldrich</td>
			<td style="width:119px;">D125407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Female athymic nude mice (Nu/Nu strain), 6&#8211;8 weeks age</td>
			<td colspan="2">Harlan (Livermore, CA)</td>
			<td></td>
		</tr>
	</tbody>
</table>

a facile and efficient approach for the production of reversible disulfide cross-linked micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

Preparation and Characterization of Drug-loaded, Disulfide Cross-linked Micelles
Amphiphilic polymer PEG5K-Cys4-Ebes8-CA8 is a dendritic polymer capable of forming a disulfide cross-linked micellar system for cancer drug delivery. Structurally, it is defined as a dendritic oligomer of cholic acids (hydrophobic domain) linked to one end of the linear PEG molecule (hydrophilic domain, molecular weight 5...

Watch this Scientific Journal Video about A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles at JoVE.com

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

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Research

JoVE Journal

Biochemistry

8.8K Views.  University of California, Davis. To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Video: A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

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 ...

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...