Name: development of recombinant proteins to treat chronic pain
Uploaded: 2018-04-11T13:30:00
Description: Watch this Scientific Journal Video about Development of Recombinant Proteins to Treat Chronic Pain at JoVE.com

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
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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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			<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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			<td height="21" style="height:21px;">GIBCO FreeStyle 293 Expression Medium</td>
			<td>Life technologies</td>
			<td>12338018</td>
			<td>Culture medium</td>
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			<td height="21" style="height:21px;">293fectin Reagent</td>
			<td style="width:143px;">Invitrogen</td>
			<td>12347019</td>
			<td>Transfection reagent</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:143px;">Sigma</td>
			<td>S7653-1kg</td>
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			<td height="21" style="height:21px;width:296px;">Sodium bicarbonate&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">31437</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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 ...

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Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...