Part of this work has been funded by a Utrecht University Life Sciences grant

All animal experiments were performed in accordance with international guidelines and prior approval from the local experimental ethical committee. Whole blood was obtained from the Mini Donor Service (Mini Donor Dienst, MDD) at the University Medical Center Utrecht (UMCU) in the Netherlands. The MDD has received positive approval from the Medical Ethics Committee of the UMCU (Medisch Ethische Toetscommissie) for the protocol number 07-125/C.
1. Protein Production and Characterization
<ol>
	...

<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=Current+perspectives+on+intrathecal+drug+delivery.">Current perspectives on intrathecal drug delivery.</a> J Pain Res. 7, 615-626 (2014).</li><li>Eijkelkamp, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Piezo2+in+EPAC1-dependent+mechanical+allodynia.">A role for Piezo2 in EPAC1-dependent mechanical allodynia.</a> Nat Commun. 4, 1682 (2013).</li><li>Bradman, M. 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The authors have nothing to disclose.

This manuscript describes methods for the production and characterization of a recombinant IL4-10 fusion protein, and methods to test its efficacy in inhibiting inflammatory hyperalgesia in mouse models of persistent inflammatory pain. The production and purification of the IL4-10 fusion protein is performed on a small scale. HEK293 cells are selected as an expression system for protein production because they enable post-translational modifications that cannot be achieved in prokaryotic expression systems. Post-translat...

Chronic pain remains one of the most debilitating and under-treated medical problems of the 21st century, affecting &#62;20% of the adult population1,2. However, treatments to provide relief from chronic pain are often ineffective or must be discontinued due to severe side effects3. Importantly, currently available drugs only provide symptomatic relief, but do not significantly modify or cure chronic pain. Although chronic pain appears to be a neurological disorder, evidence suggests involvement of the immune system in chronic pain development4,5. Moreover, immune-based approaches to treat pain are emerging. For example, anti-inflammatory cytokines inhibit pain in several models of chronic pain6,7,8. However, anti-inflammatory cytokines have a short half-life, reducing their potential pain-inhibiting effects. Moreover, anti-inflammatory cytokines work most optimally in concert with each other. To overcome these limitations, we recently fused the anti-inflammatory cytokines interleukin-4 (IL4) and interleukin-10 (IL10) into one molecule. The IL4-10 fusion protein shows superior efficacy in inhibiting chronic inflammatory and neuropathic pain compared to the individual cytokines9. Here we describe how such fusion protein is produced, purified, and how its quality is controlled.
IL4-10 fusion protein is produced in human cells by transient transfection of HEK293-F cells with a pUPE expression vector carrying the cDNA sequence coding the IL4-10 fusion protein. HEK293-F cells are chosen to allow for post-translational modification of the protein, something that does not occur in bacterial expression systems. To optimize glycan capping with sialic acid, cDNA coding beta-galactoside-2, 3-sialyl-transferase is incorporated in the vector as a second transgene. The fusion protein is purified using affinity protein purification of the culture supernatant because it is more powerful than purification by other methods e.g. size-exclusion or ion exchange chromatography10,11. To purify the IL4-10 fusion protein, we used in-house made monoclonal antibodies against IL4. Evaluation of the purity and bioactivity of purified IL4-10 fusion protein is performed as a part of quality control. The purity of produced batches is evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and High Pressure Size Exclusion Chromatography (HP-SEC). The bioactivity of the IL4-10 fusion protein is evaluated by measuring its capacity to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF&#945;) production in whole blood cultures, and comparing it to the combination of the individual cytokines.
Finally, in order to test the capacity of IL4-10 fusion proteins to inhibit chronic pain, we describe how the fusion protein can be tested as analgesic in widely used mouse models of persistent inflammatory pain12,13,14. Here we describe the methods of an inflammatory pain model. However, it is important to note that other pain models can be used (e.g., neuropathic pain models), depending on the research questions that need to be answered. To assess pain in these models, it is important to use a variety of behavioral measures that include evoked and non-evoked pain measures. Here, we described the methods of assessment for changes in mechanically and thermally evoked behavioral responses. Mechanical sensitivity to innocuous stimuli is assessed using the Von Frey test, whilst thermal sensitivity is assessed using the Hargreaves test. Importantly, non-evoked hyperalgesia/allodynia is measured using the Dynamic Weight Bearing test. These measures are widely accepted as pain measures and yield important information on pain thresholds and potential pain experienced by the animals15,16,17. Other measures to assess non-evoked pain (e.g., stimulus independent), such as the conditioned place preference test, may be valuable18. To assess the potential of the drug to inhibit pain, we performed intrathecal administration of the fusion protein, as with this route of administration less protein dose is needed to reach pain-related areas and avoid systemic (side-) effects19,20.

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		<tr height="21">
			<td height="21" style="height:21px;width:296px;">FreeStyle 293-F cells</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:161px;">R790-07</td>
			<td style="width:269px;">Human embryonic kidney cells&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">GIBCO FreeStyle 293 Expression Medium</td>
			<td>Life technologies</td>
			<td>12338018</td>
			<td>Culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">293fectin Reagent</td>
			<td style="width:143px;">Invitrogen</td>
			<td>12347019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO Opti-MEM + GlutaMAX</td>
			<td>Life technologies</td>
			<td>51985026</td>
			<td>Reduced Serum Medium for use during cationic lipid transfections&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">GIBCO RPMI Medium 1640 (1x)</td>
			<td>Life technologies</td>
			<td style="width:161px;">52400-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CNBr-Activated Sepharose 4B</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>17-0430-01</td>
			<td>pre-activated media for coupling antibodies or other large proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hydrochloric acid fuming 37%&#160;</td>
			<td style="width:143px;">Merck</td>
			<td>1003171000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:143px;">Sigma</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium bicarbonate&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">31437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Trizma hydrochloride</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">T3253-500G</td>
			<td>TRIS hydrochloride&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic Acid 100%&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Merck</td>
			<td style="width:161px;">1.00063.1000&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glycin-HCl&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">G2879&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Pharmacie, UMCU</td>
			<td style="width:161px;"></td>
			<td>Phosphate-Buffered Saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10X TGS</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">161-0772</td>
			<td>Tris/Glycine/SDS Buffer for SDS electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Mini-PROTEAN TGX Gels</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">456-1046</td>
			<td>12% SDS precast gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Trans-Blot Turbo Transfer Pack</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">170-4157</td>
			<td>Western blot transfer packs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yarra 3u SEC-2000 column</td>
			<td style="width:143px;">Phenomenex</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstantBlue Protein Stain</td>
			<td style="width:143px;">Expedeon</td>
			<td>ISB1L</td>
			<td>ready to use Coomassie protein stain for polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td>Human IL-10 DuoSet ELISA</td>
			<td>R&#38;D</td>
			<td>DY217B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Human TNF&#945; ELISA Set</td>
			<td>Diaclone</td>
			<td>851570020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>BCA Pierce Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carrageenan</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">22049</td>
			<td>plant mucopolysaccharide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CFA</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">F5881</td>
			<td>vaccine adjuvant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hamilton syringe</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">20779</td>
			<td>glass syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Animal Enclosure</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">433</td>
			<td style="width:269px;">Animal Enclosure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Von Frey mesh stand</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">410</td>
			<td>Mesh Stand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">von Frey hairs&#160;</td>
			<td style="width:143px;">Stoelting</td>
			<td>58011</td>
			<td>touch test sensory probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Plantar Test (Hargreaves Method)</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">390G</td>
			<td>plantar test with heated glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynamic Weight Bearing test</td>
			<td style="width:143px;">Bioseb</td>
			<td style="width:161px;">BIO-DWB-AUTO-M</td>
			<td>postural deficit test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Econo-Column Columns, 1.5 &#215; 30 cm</td>
			<td style="width:143px;">BIO-RAD</td>
			<td>7371532</td>
			<td>glass chromatography column&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SnakeSkin Dialysis Tubing&#160;</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:161px;">88242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Minisart NML Syringe Filter&#160;</td>
			<td style="width:143px;">Sartorius</td>
			<td style="width:161px;">16555-K</td>
			<td>single use filter unit, 0.45 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY Cell Counter and Analyzer</td>
			<td style="width:143px;">Roche</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of recombinant proteins to treat chronic pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

A representative picture of an SDS-PAGE gel containing different fractions obtained during affinity chromatography purification is shown in Figure 1A. In the load (L) and flow through (FT) fractions, all proteins present in the HEK293 supernatant are observed. No protein is observed in the wash (W) fraction. In the elution (E) fraction, two bands of 35 and 37 kDa are observed corresponding to two different glycoforms of IL4-10 fusion protein ...

Watch this Scientific Journal Video about Development of Recombinant Proteins to Treat Chronic Pain at JoVE.com

Development of Recombinant Proteins to Treat Chronic Pain

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

The overall goal of these procedures is to produce a fusion protein of the anti-inflammatory cytokines IL4 and IL10, control its quality, and evaluate its functional activity and analgesic properties in mirroring models of chronic inflammatory pain. This detailed methods aid in developing novel biologics and test their quality and functionality. The advantage of these techniques is that you can produce a sufficient amount of recombinant fusion proteins, which enable its assessment for its analgesic properties in vivo.To evaluate the therapeutic potential of the new developed fusion protein, it is important to have a well-characterized batch and to use a variety of behavioral tests. The implication of these methods is that they provide insight in highly needed new therapies for chronic pain but also extend the potential towards other inflammatory diseases. Generally, scientists new to these methods will struggle to be able to perform all procedures shown here, since expertise in different fields is required.After subculturing 100 milliliters of HEK 293F cells in 500-milliliter Erlenmeyer flasks according to the text protocol, add 6.6 milliliters of DNA transfection reagent mixture to each flask of cells. Culture the cells at 37 degrees Celsius, 8%CO2, and 95%humidity on an orbital shaker rotating at 125 rpm for three days. Then, harvest the culture supernatant containing secreted IL4-10 fusion protein.To carry out protein purification, pass 100 milliliters of 10-fold concentrated cell culture supernatant through a previously prepared, IL4-coupled Sepharose bead column at a flow rate of one milliliter per minute. Use 50 milliliters of PBS to wash the column. Then, apply 12.5 milliliters of 0.1 molar glycine buffer to elute the bound IL4-10 fusion protein into 2.5 milliliter fractions at a flow rate of one milliliter per minute.Next, add 350 microliters of one molar Tris buffer, pH 9, to each fraction to bring the pH to 7. Use dialysis tubing with a 16-millimeter dry diameter and a 3.5K molecular weight cut-off to dialyze the neutralized fractions against two liters of PBS overnight. Then, use disposable filters with 0.45-micrometer pore diameter to filter sterilize the dialyzed fractions.Aliquot the sterile IL4-10 fusion protein solution and store it at negative 80 degrees Celsius. Evaluate the purity of eluted IL4-10 fusion protein by first running a sample on a 12%SDS-PAGE gel and stain the gel with Coomassie blue. Then, carry out high-pressure size-exclusion chromatography by loading 40 microliters of one milligram per milliliter protein onto the column.Determine the concentration of each batch of purified IL4-10 fusion protein using an IL10 ELISA and a BCA protein assay kit according to the manufacturer's protocols. Use RPMI medium to prepare three-fold serial predilutions of the purified IL4-10 fusion protein over a concentration range of five to 1, 215 nanograms per milliliter. Into the wells of a 48-well plate, add 50 microliters of IL4-10 fusion protein, 75 microliters of RPMI medium, 25 microliters of LPS, and 100 microliters of human blood.Incubate the plate at 37 degrees Celsius, 8%CO2, and 95%humidity for 18 hours. Then, use a TNF-alpha ELISA according to the manufacturer's protocol to evaluate the concentration of TNF-alpha in culture supernatants. Maintain prepared carrageenan or CFA solutions in syringes with 30-gauge needles at room temperature for at least 15 minutes before injection.Then, into the thumb of the hind paw of a non-anesthetized mouse, insert the needle approximately 0.5 centimeters, directing it towards the heel and inject 20 microliters of the compound. To carry out pain measurements using the Von Frey test, place an animal into an acrylic cage on a wire-mesh stand and allow the mouse to acclimatize for at least 15 minutes. Apply the Von Frey filaments ranging from 0.02 to four grams perpendicular to the paw surface with sufficient force to cause slight bending against the skin and hold them for approximately three seconds.Measure the paw withdrawal threshold in response to each hair, considering any movement of the paw away from the applied hair as a positive withdrawal response. Place the animals into cages on a pre-warmed glass plate and allow the mice to acclimate for at least 15 minutes prior to measurements. Determine the heat withdrawal latency times by using a Hargreaves apparatus to heat the paw of the animals with a focused visible light beam.Before the dynamic weight-bearing test, measure the body weight of each animal. Place an animal in a floor-instrumented dynamic weight-bearing system assembled onto a cover supporting a camera that will record mouse movements. Allow the animal to acclimate for 0.5 to one minute before recording mouse movements for five minutes.Then, carry out analysis of each video, ensuring that each limb is recognized correctly by the software at the timeframe indicated by the software. Backfill the injectable compounds into a clean 25-microliter glass Hamilton syringe connected to a 27-gauge needle. Place the mouse on the table with its head placed in the nosecone of the apparatus and maintain the mouse sedation with 1.5 to 2%isoflurane with an oxygen flow rate of two liters per minute.Check that the mouse is properly anesthetized by pinching the paw and ensuring that it is nonresponsive. Hold the mouse firmly by spine posterior to the ribs. Position the needle vertically between the L5 and L6 lumbar vertebrae and carefully insert it through the skin into the intervertebral space.Confirm correct targeting by verifying that a tail flick occurred. If only a single paw flick is observed, the needle is likely not placed correctly. Then, slowly inject five microliters of the compound, wait a couple of seconds, and slowly retract the needle.A representative picture of an SDS-PAGE gel containing affinity chromatography fractions is shown here. Two bands of 35 and 37-kilodaltons are observed in the elution fraction corresponding to two different glycoforms of IL4-10 fusion protein. This figure illustrates the results of a potency assay in a whole-blood assay of two different IL4-10 fusion protein batches.A dose-dependent inhibition of LPS-induced TNF-alpha release is observed, showing comparable bioactivity for the two IL4-10 fusion protein batches. In these experiments, persistent inflammatory pain was induced by intraplantar injection of 2%carrageenan. On day six after induction, mice were injected intrathecally with IL4-10 fusion protein.IL4-10 significantly resolved both mechanical and thermal hyperalgesia and reached its maximal effect 24 hours after injection. Here, inflammatory pain was induced by an intraplantar injection of 20 microliters of CFA. Weight-bearing on each paw was measured using the dynamic weight-bearing device.These results show that intraplantar CFA injection reduced weight-bearing of the affected paw. Seven days after intraplantar CFA injection, intrathecal injection of IL4-10 fusion protein was carried out. Two days later, the reduction in weight-bearing of the affected paw was attenuated compared to vehicle-injected mice.Once mastered, using these procedures, three milligram of fusion protein can be obtained from every liter of cultured cells. It is good to keep in mind to choose expression system compatible with future GMP-production of fusion proteins. To test the analgesic potential of this novel fusion protein, other models of chronic pain can be used, like nerve injury or chemotherapy-induced neuropathy.A variety of behavioral tests is shown in this video, but other pain tests suggest conditioned place preference could also be performed. Don't forget that intrathecal injections require well-trained personnel and that the injection volume is rather limited. After watching this video, you should have a good understanding of how to produce a fusion protein and test its efficacy in vivo in various pain models.

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9.4K vistas.  University Medical Center Utrecht, Utrecht University. The overall goal of these procedures is to produce a fusion protein of the anti-inflammatory cytokines IL4 and IL10, control its quality, and evaluate its functional activity and analgesic properties in mirroring models of chronic inflammatory pain. This detailed methods aid in developing novel biologics and test their quality and functionality. The advantage of these techniques is that you can produce a sufficient amount of recombinant fusion proteins, which enable its assessment for its ana...