Name: efficient and scalable production of full-length human huntingtin variants in mammalian cells using a transient expression system
Uploaded: 2021-12-10T14:15:00
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Description: Watch this Scientific Journal Video about Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System at JoVE.com

We thank the Department of Pharmaceutical Sciences, The State University of New York at Buffalo, for performing MS analysis of HTT. This work was a collaborative effort with the CHDI Foundation. We specifically thank Elizabeth M. Doherty; Ignacio Munoz-Sanjuan; Douglas Macdonald, CHDI Foundation; and Rory Curtis, Curia, for their invaluable input during the preparation of this manuscript. We are also grateful to Michele Luche, Mithra Mahmoudi, and Stephanie Fox for their support of this research effort.

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The authors declare that they have no conflicts of interest with the contents of this article.

We describe here a transient transfection, expression, and purification method to generate multiple FL HTT protein constructs with suitable purity and homogeneity for use as standards for immunoassay and MS assay development, controls for western blot analysis, and for structure-function studies. This transient expression method is scalable and versatile and enables the user to generate low-milligram quantities of FL HTT variants more efficiently than using stable cell lines or virus-based methods described previously21,22,23,24. Routinely, 2-5 mg of highly purified FL HTT can be generated from a 2 L scale protein production run in less than a week using the transient expression method once the plasmid is constructed, with a typical yield of 1-2.5 mg of FL HTT per liter of cell culture.
The transient expression method described here overcomes many obstacles in stable cell line expression, such as the long time needed to establish cell lines and difficulties in storage and maintaining stable cell lines. PEI is also relatively inexpensive compared to other transfection reagents in the market, making large-scale transfection economically viable. There are also limitations in the protocol: transfection efficiency largely depends on the quality of the plasmids, optimal cell growth, and how well PEI is stored and prepared. Operators need to take special care and perform quality controls in those critical steps to avoid a drastic drop in protein yields. Anti-FLAG resin used in the protocol is also relatively expensive and shows reduced capture of FL HTT after several purifications and regenerations. Some researchers may find it more practical to switch to a different tag to allow more robust regeneration of the affinity resin.
Various cell lines and expression conditions were tested to optimize FL HTT expression levels. HEK293 cells were chosen for the expression of FL HTT because of the high expression of protein and the ease of handling in a suspension culture format, making the method suitable for large-scale expression in either shakers or bioreactors. A higher FL HTT protein expression level can be achieved at lower culturing temperatures such as 32 &#176;C rather than using the customary temperature of 37 &#176;C. It is possible that the lower temperature may slow protein synthesis and promote correct folding of FL HTT40. However, this phenomenon is not specific to FL HTT or the cell lines tested. The reduced posttransfection temperature has been widely used in pharmaceutical protein expression in CHO cells. Although the mechanism is not fully understood, it is thought that low temperatures arrest the cell cycle in the G1 phase and divert cellular energy to protein production41.
Full-length HTT purified from mammalian cells co-elutes with the chaperone Hsp7024, and Mg-ATP washing steps can remove the Hsp70 protein. Interestingly, co-eluted Hsp70 is not observed in FL HTT purified from an insect cell expression system21,22,23. This may reflect a difference in the PTMs of FL HTT or heat shock protein responses to the overexpression of FL HTT in mammalian and insect cells. Once the recombinant protein has been stripped of Hsp70, non-ionic detergents such as CHAPS or DDM are required to stabilize the monomeric form of FL HTT.
The oligomerization states of FL HTT variants were analyzed using Blue Native PAGE and SEC-MALS. A small fraction of dimeric and higher-order oligomeric HTT was present when analyzed by either Blue Native PAGE or SEC-MALS. Of note, higher-ordered oligomers formed by FL HTT do not appear to correlate with polyQ length, and even the Exon1 deletion mutant displays a similar oligomer-dimer-monomer ratio. The actual variations in oligomer content among these constructs are likely due to minor differences in the production and handling of each batch. In contrast to aggregates and fibrils formed by HTT Exon140,41, the higher-ordered oligomers of FL HTT remained soluble and could be analyzed by SEC and Native PAGE.
Purified monomeric FL HTT is only relatively stable. Prolonged storage at 4 &#176;C, short incubations at room temperature, or concentrations &#62; 1 mg/mL will all convert monomeric FL HTT to dimeric and higher-ordered oligomeric forms even though there is no visible precipitation observed under those conditions. Purified monomeric FL HTT maintained at &#8804;1 mg/mL remained relatively stable at -80 &#176;C in storage buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 5% v/v glycerol, 0.5% w/v CHAPS, and 5 mM DTT) as previously described24. Up to 6 freeze-thaw cycles of FL HTT prepared and stored in this manner did not cause visible precipitation of the protein, although a slight shift to a higher oligomeric state was observed by SEC-MALS (Supplemental Figure S2). Samples were also analyzed by SDS PAGE following repeated freeze-thaw cycles. No visible precipitates were observed; no aggregates or additional degradation products were seen by SDS-PAGE (Supplemental Figure S3). The long-term stability of purified FL HTT is still under investigation. In the absence of conclusive long-term data, we recommend storing purified FL HTT at -80 &#176;C for no longer than 6 months.
High-quality, recombinant FL HTT protein variants and the methods to produce them are in high demand by the HD research community. These proteins are in use as immunoassay and MS analytical standards, in structural studies, and for the development of novel FL HTT-specific assays. The large-scale transient expression methods described here have consistently produced milligram quantities of FL HTT variants with &#62;95% purity, providing essential tools for HTT studies. Production of tens of milligrams of highly purified FL HTT polyQ variants and other mutants in support of HD research has become routine.

Huntington&#39;s disease (HD) is a rare neurodegenerative disease primarily characterized by unsteady and involuntary motor movement, as well as cognitive and psychiatric alterations, such as personality changes and apathy1,2. HD is associated with an expansion of the CAG repeat tract located in exon 1 of the huntingtin gene (HTT) to more than 35 repeats, with a higher number of CAG repeats correlating with an earlier onset of the disease3,4. The translational product of HTT, the huntingtin protein (HTT), is implicated in neuronal viability and brain development5,6,7,8,9.
HTT is a scaffolding protein reported to participate in a wide range of cellular processes, vesicle transport, cell division, ciliogenesis, and autophagy10,11. However, the molecular pathogenesis of HD is not entirely clear, and the identification of key protein interactors mediating the pathological impact of polyQ-expanded mHTT is lacking. Some research suggests a gain of toxic function from mHTT driven by the oligomerization propensity of the expanded HTT protein, as HTT aggregates have been identified in neurons and glia in HD patients and animal models of the disease12,13,14,15,16,17. To fuel the investigation of the function and structure of FL HTT and mHTT variants and supply researchers with high-quality protein standards for assay development, a robust and scalable supply of homogenous recombinant protein is needed.
Due to its size (aa 1-3,144, numbering based on polyQ length Q23), proteolytic instability, and propensity to aggregate, FL HTT has proven difficult to express and isolate as a soluble protein. Previously, the exon 1 region (aa 2-90) of HTT has been expressed and purified at a large scale using various tags that can increase the solubility of the protein in Escherichia coli18,19,20. FL HTT was first expressed and purified in an insect cell expression system using baculovirus21,22, and low-resolution 30 &#197; electron microscopy (EM) structures of chemically crosslinked FL Q23-HTT and Q78-HTT were reported23. The investigation of HTT structure was further advanced when the production of FL Q17, Q46, and Q128-HTT with native posttranslational modifications (PTMs) was achieved in human cells using stable cell lines or adenovirus expression systems24. These studies suggest that although purified HTT mainly exists in the monomeric state, it also tends to form high-order oligomers and aggregates.
Analytical ultracentrifugation of FL Q128-HTT, with a highly expanded polyQ region, afforded more oligomeric and aggregate fractions than the protein with the non-expanded polyQ region24. Using a stable cell line, a strategy has been successfully adapted to stabilize FL HTT by co-expression with the interaction partner HAP40. A cryo-EM structure of the FL HTT and HAP40 complex has been solved at an average 4 &#197; resolution using the purified protein complex (PDB:6EZ8)25. This co-expression strategy has been adapted successfully to a baculovirus system, and a series of high-quality HTT variants with different polyQ lengths have been expressed and purified from insect cells26. Since then, more cryo-EM structures of the complex of HTT with variable polyQ lengths and HAP40 and higher resolution structures were solved and deposited in the Protein Data Base27,28 (PDB: 7DXK, 7DXH, 6X9O).
We optimized a transfection and expression method in HEK293 cells, using polyethylenimine (PEI), for rapid transient expression of FL HTT. As a proof-of-principle, FL HTT variants containing 23 glutamines (FL Q23-HTT) were first purified and characterized using a modification of a purification method described previously24. This transient transfection method is convenient, highly efficient, and scalable; it can produce purified HTT with yields of 1-2 mg/L, comparable to the stable cell line method reported24. Because the protein is produced in a human cell line, the HTT produced is more likely to have native human PTMs when subjected to mass spectrometry proteomics analysis11,29,30,31. Milligram quantities of the FL Q48-HTT, FL Q73-HTT, and exon1-deleted (&#916;Exon1-HTT) variants of FL HTT were produced, demonstrating that the transient expression method is especially useful in rapidly producing alternative variants of HTT without depending on the time-consuming effort required to establish stable cell lines for production.
The following protocol exemplifies the standard method used in these authors&#39; laboratory for cell culture, transfection, protein purification, and postpurification protein characterization to produce FL Q23-HTT from a 2 L cell culture. The protocol can be scaled up to larger cultures or adapted to purify other HTT variants. Up to 10 L cell cultures of FL HTT and various site or truncation mutations of HTT and HTT homologs have been performed successfully in the laboratory using the same protocol. Purified FL HTT contains a high percentage of monomers along with dimers and higher-order oligomers. The same aggregate profile is observed among the variants produced (Q23, Q48, Q73, and deleted Exon1). As aggregation can occur when proper care is not taken, a formulation and freeze-thaw stability study was conducted to identify the best conditions for protein handling. Methods, such as Blue Native PAGE and SEC/MALS-RI, are also described to analyze the HTT oligomer content as part of the quality control process. To benefit the HD research community, plasmids and HTT proteins described in this study are also deposited in the HD Community Repository at the Coriell Institute (www.coriell.org/1/CHDI).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 kDa concentrator-Amicon</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td>Protocol Section Number-6.2.4</td>
		</tr>
		<tr>
			<td>20x blue native PAGE running buffer</td>
			<td>Invitrogen</td>
			<td>BN2001</td>
			<td>Protocol Section Number-8.1</td>
		</tr>
		<tr>
			<td>20x TBS</td>
			<td>Thermo Fisher</td>
			<td>PI28358</td>
			<td>Protocol Section Number-5.1</td>
		</tr>
		<tr>
			<td>4x blue native PAGE sample buffer</td>
			<td>Invitrogen</td>
			<td>BN2003</td>
			<td>Protocol Section Number-8.3</td>
		</tr>
		<tr>
			<td>4x LDS loading buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td>Protocol Section Number-5.3</td>
		</tr>
		<tr>
			<td>5 L Erlenmeyer flasks</td>
			<td>Corning</td>
			<td>431685</td>
			<td>Protocol Section Number-4.2</td>
		</tr>
		<tr>
			<td>Agarose gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Protocol Section Number-2.2</td>
		</tr>
		<tr>
			<td>Anti-clumping agent</td>
			<td>Thermo Fisher</td>
			<td>0010057AE</td>
			<td>Protocol Section Number-4.8</td>
		</tr>
		<tr>
			<td>anti-FLAG M2 affinity gel</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td>Protocol Section Number-6.1.1</td>
		</tr>
		<tr>
			<td>anti-FLAG M2</td>
			<td>Sigma</td>
			<td>F3165</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>Anti foam-Excell anti foam</td>
			<td>Sigma</td>
			<td>59920C-1B</td>
			<td>Protocol Section Number-4.8</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A6419</td>
			<td>Protocol Section Number-6.1.4.4</td>
		</tr>
		<tr>
			<td>BEH 450 SEC</td>
			<td>Waters</td>
			<td>186006851</td>
			<td>2.5 &#181;m x 4.6 mm x 150 mm 
			Protocol Section Number-7.3</td>
		</tr>
		<tr>
			<td>blue native PAGE 5% G-250 sample additive</td>
			<td>Invitrogen</td>
			<td>BN2004</td>
			<td>Protocol Section Number-8.3</td>
		</tr>
		<tr>
			<td>carbenicillin</td>
			<td>Thermo Fisher</td>
			<td>10177012</td>
			<td>Protocol Section Number-2.5</td>
		</tr>
		<tr>
			<td>centrifuge - Sorvall Lynx 6000</td>
			<td>Thermo Fisher</td>
			<td>75006590</td>
			<td>Protocol Section Number-6.1.3</td>
		</tr>
		<tr>
			<td>Cell Counter - ViCELL</td>
			<td>BECKMAN COULTER</td>
			<td></td>
			<td>Protocol Section Number-4.3</td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Anatrace</td>
			<td>C316S</td>
			<td>Protocol Section Number-6.1.4.6</td>
		</tr>
		<tr>
			<td>Competent E. coli cells-TOP10</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td>Protocol Section Number-2.4</td>
		</tr>
		<tr>
			<td>digitonin</td>
			<td>Sigma</td>
			<td>D141</td>
			<td>Protocol Section Number-5.1</td>
		</tr>
		<tr>
			<td>differential refractive index detector</td>
			<td>Wyatt</td>
			<td></td>
			<td>Protocol Section Number-7.1</td>
		</tr>
		<tr>
			<td>DYKDDDDK peptide</td>
			<td>Genscript</td>
			<td></td>
			<td>Peptide synthesis service 
			Protocol Section Number-6.1.4.6</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS</td>
			<td>Protocol Section Number-5.1</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Giga Kit</td>
			<td>Qiagen</td>
			<td>12391</td>
			<td>Protocol Section Number-3.3</td>
		</tr>
		<tr>
			<td>Endotoxin free water</td>
			<td>Cytiva</td>
			<td>SH30529.03</td>
			<td>Protocol Section Number-4.1</td>
		</tr>
		<tr>
			<td>endotoxin quantification kit-CRL Endosafe Nexgen-PTS detection system</td>
			<td>Charles River</td>
			<td>PTS150K</td>
			<td>Protocol Section Number-3.4</td>
		</tr>
		<tr>
			<td>fixed angle rotor A23-6x100 rotor</td>
			<td>Thermo Fisher</td>
			<td>75003006</td>
			<td>Protocol Section Number-6.1.3</td>
		</tr>
		<tr>
			<td>FPLC software- Unicorn 6.2</td>
			<td>Cytiva</td>
			<td></td>
			<td>Protocol Section Number-6.1.4</td>
		</tr>
		<tr>
			<td>Gene synthesis</td>
			<td>Genscript</td>
			<td></td>
			<td>Gene synthesis service 
			Protocol Section Number-1.2</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Honeywell</td>
			<td>60-048-023</td>
			<td>Protocol Section Number-5.6</td>
		</tr>
		<tr>
			<td>Growth Medium-Expi293 expression medium</td>
			<td>Thermo Fisher</td>
			<td>A1435102</td>
			<td>Protocol Section Number-4.2</td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>Thermo Fisher</td>
			<td>R79007</td>
			<td>Protocol Section Number-4</td>
		</tr>
		<tr>
			<td>high shear homogenizer-Microfluidizer</td>
			<td>MicroFluidics</td>
			<td>LM10</td>
			<td>Protocol Section Number-6.1.3</td>
		</tr>
		<tr>
			<td>HPLC - 1260 infinity II Bio-Insert HPLC</td>
			<td>Agilent</td>
			<td></td>
			<td>Protocol Section Number-7.1</td>
		</tr>
		<tr>
			<td>Image Studio</td>
			<td>LiCor</td>
			<td></td>
			<td>Image analysis software 
			Protocol Section Number-5.1</td>
		</tr>
		<tr>
			<td>MAB2166</td>
			<td>Sigma</td>
			<td>MAB2166</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB2168</td>
			<td>EMD</td>
			<td>MAB2168</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB3E10</td>
			<td>Santa Cruz</td>
			<td>SC-47757</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB4E10</td>
			<td>Santa Cruz</td>
			<td>SC-7757</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB5490</td>
			<td>Sigma</td>
			<td>MAB5490</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB5492</td>
			<td>Sigma</td>
			<td>MAB5492</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>MAB8A4</td>
			<td>Santa Cruz</td>
			<td>SC-47759</td>
			<td>Protocol Section Number-5.7</td>
		</tr>
		<tr>
			<td>multi-angle light scattering detector</td>
			<td>Wyatt</td>
			<td></td>
			<td>Protocol Section Number-7.1</td>
		</tr>
		<tr>
			<td>NativeMark Unstained Protein Standard</td>
			<td>Invitrogen</td>
			<td>LC0725</td>
			<td>Protocol Section Number-8.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>Protocol Section Number-5.6</td>
		</tr>
		<tr>
			<td>NheI</td>
			<td>New England Biolab</td>
			<td>R0131S</td>
			<td>Hi-Fi version available 
			Protocol Section Number-2.2</td>
		</tr>
		<tr>
			<td>NuPAGE 3&#8211;8% Tris acetate gels</td>
			<td>Thermo Fisher</td>
			<td>EA0375PK2</td>
			<td>Protocol Section Number-5.4</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running buffer</td>
			<td>Invitrogen</td>
			<td>LA0041</td>
			<td>Protocol Section Number-5.4</td>
		</tr>
		<tr>
			<td>PEI 25K</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td>Protocol Section Number-4.1</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15070063</td>
			<td>Protocol Section Number-4.2</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Cytiva</td>
			<td>SH30256.02</td>
			<td>Protocol Section Number-4.5</td>
		</tr>
		<tr>
			<td>plasmid miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>Protocol Section Number-2.6</td>
		</tr>
		<tr>
			<td>PmeI</td>
			<td>New England Biolab</td>
			<td>R0560S</td>
			<td>Protocol Section Number-2.2</td>
		</tr>
		<tr>
			<td>precast Bis-tris gel- 3-12% NativePAGE Novex Bis-Tris Gel</td>
			<td>Invitrogen</td>
			<td>BN1003BOX</td>
			<td>Protocol Section Number-8.4</td>
		</tr>
		<tr>
			<td>protease inhibitor cocktail</td>
			<td>GoldBio</td>
			<td>GB-331-1</td>
			<td>Protocol Section Number-5.1</td>
		</tr>
		<tr>
			<td>SEC-MALS analysis software - Astra 7</td>
			<td>Wyatt Technology</td>
			<td></td>
			<td>Protocol Section Number-7.6</td>
		</tr>
		<tr>
			<td>secondary antibody -IRdye 800 CW goat anti-mouse IgG</td>
			<td>LiCor</td>
			<td>926-32210</td>
			<td>Protocol Section Number-5.9</td>
		</tr>
		<tr>
			<td>Superose 6 pg XK 16/70</td>
			<td>Cytiva</td>
			<td>90100042</td>
			<td>Protocol Section Number-6.2</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td>Protocol Section Number-5.6</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Thermo Fisher</td>
			<td>AAJ20605AP</td>
			<td>Protocol Section Number-6.1.1</td>
		</tr>
		<tr>
			<td>UV spectrometer - Nanodrop 8000</td>
			<td>Thermo Fisher</td>
			<td>ND-8000-GL</td>
			<td>Protocol Section Number-2.2</td>
		</tr>
		<tr>
			<td>XK26/100</td>
			<td>Cytiva</td>
			<td>28988951</td>
			<td>Protocol Section Number-6.1.1</td>
		</tr>
	</tbody>
</table>

efficient and scalable production of full-length human huntingtin variants in mammalian cells using a transient expression system

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

Watch this Scientific Journal Video about Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System at JoVE.com

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

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