Name: generation of native, untagged huntingtin exon1 monomer and fibrils using a sumo fusion strategy
Uploaded: 2018-06-27T13:30:00.000+00:00
Duration: 11 min 22 s
Description: Watch this Scientific Journal Video about Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy at JoVE.com

This work was funded primarily by grants from the CHDI foundation and the Swiss National Science Foundation. We thank Dr. Sophie Vieweg for useful discussions during the development of this new expression system and other members of the Lashuel group for sharing their experience with this expression system and for their input and valuable feedback. We also thank Prof. Oliver Hantschel for providing the ULP1 plasmid. The authors thank Dr. John B. Warner and Dr. Senthil K. Thangaraj for critical review of the manuscript

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

In this protocol, we have outlined an efficient procedure for obtaining milligram quantities of native, untagged Httex1 containing 23 or 43 glutamine residues. This was achieved by expressing Httex1 as a C-terminal fusion to a His6-SUMO tag, which is used to isolate the fusion protein from the cell lysate by IMAC and is cleaved prior to HPLC purification of Httex1. While the SUMO strategy has been used in the production of several other proteins, our method shows that the unique properties SUMO could also be used to generate intrinsically disordered, aggregation-prone, amyloidogenic protein that have previously proved to be extremely difficult to handle and produce43,44. We present a protocol that is straightforward, easy to use and comparable to a protocol for the generation of a &#34;well-behaved&#34; protein. The SUMO fusion solubilizes and stabilizes Httex1 during expression and the IMAC purification step. Premature cleavage of the tag, as observed with the intein strategy10 and aggregation were no longer an issue.
Intrinsically disordered proteins are especially vulnerable to degradation. While N-terminal degradation in the N17 region is not an issue using this protocol, truncations in the PRD of Httex1 can occur. As the truncated proteins are very similar to Httex1 in hydrophobicity, charge and size, removing them by chromatographic means is challenging, thus it is best to prevent their formation in the first place. Sticking closely to the protocol, always working on ice and using a sufficient amount of protease inhibitor should help keep the level of observed truncation very low. Applying a fusion tag at the C-terminus of Httex1 could remove truncations in the PRD easily as the truncated protein would lose the affinity tag as well. However, if the native sequence needs to be maintained this option cannot be applied as Httex1 ends with proline and to the best of our knowledge there are no C-terminal fusion tags that are known to induce traceless and efficient cleavage after proline.
The most critical part of the protocol is the handling of the Httex1 liberated after cleavage of the SUMO tag by ULP1. The protein should be purified immediately by RP-HPLC. Fortunately, this is an efficient and fast reaction that is usually completed in 10-20 min at 4 &#176;C. In contrast, the intein strategy required several hours for complete cleavage of the intein, thus requiring a trade-off between incomplete cleavage and beginning aggregation in order to maximize the yield. A fast workup is required for mutant Httex1, as it will start to aggregate at the comparatively high concentration present in the cleavage reaction, whereas the 23Q variant is stable for a longer time. During the RP-HPLC purification, another advantage of SUMO becomes apparent: While the Ssp DnaB intein is hydrophobic and sticks strongly to the column, SUMO is more hydrophilic and elutes completely from the C4 reversed-phase column. Although commercial ULP1 is quite costly, the protein can be easily produced in high yield following previously published protocols29.
The critical importance of applying a disaggregation protocol prior to using Httex1 cannot be stressed enough. Lyophilized polyQ proteins such as Httex1 are stable and can be stored long periods, but are not completely soluble in water and buffers. The presence of preformed oligomers or fibrils could have a significant impact on aggregation kinetics and biophysical properties of the protein45. The disaggregation protocol described here allows the disaggregation of the protein, removal of preformed aggregates and generation of a solution of monomeric Httex1 from a lyophilized sample. We observed similar aggregation kinetics and fibril morphology for Httex1 obtained with the SUMO and the intein strategy.
Compared to previous methods for producing Httex1, the SUMO strategy described here offers several advantages and expands the range of possible studies to investigate the structure and functional properties of this protein in health and disease. The SUMO-Httex1 fusion protein is easy to handle, it can be frozen and stored or kept in solution for 24 h at ambient temperature, while the free mHttex1 would aggregate quickly. The stability and high solubility of the SUMO-Httex1 fusion proteins provide greater flexibility to manipulate the protein and/or introduce enzymatic and chemical modifications into mHttex1 that would otherwise not be possible after cleavage. This includes the introduction of post-translational modifications, fluorophores, spin labels, biotin tags, etc. The advances presented here should 1) facilitate future studies to elucidate structure-function relationships of Httex1; 2) generate new tools to investigate Htt aggregation and pathology spreading; 3) enable the development of new assays to identify molecules that stabilize mutant Httex1 and prevent its aggregation; and 4) encourage scientists from other fields to bring to work on this protein and join our quest to find cures for Huntington&#39;s disease.

Htt is a 348 kDa protein and has been implicated in several physiological functions1. When Htt contains an expanded polyQ region of more than 36 residues in its N-terminus, it causes HD2,3. HD pathology is characterized by cellular inclusions in the striatum and cortex, which leads to neuronal death and atrophy of the affected tissues4,5. Several N-terminal Htt fragments that contain the polyQ repeat tract have been detected in post-mortem brains from HD patients and are thought to be generated by proteolytic processing of the huntingtin protein6. Recent studies suggest that Httex1 could also be formed due to aberrant mRNA splicing. Httex1 contains the pathological polyQ mutation and its overexpression in animals can recapitulate many of the key features of HD7, thus highlighting a possible central role of this fragment in HD pathology and disease progression6,8,9.
Due to the high aggregation propensity of mutant Httex1 (mHttex1) with expanded polyQ tract, the majority of existing expression systems are based on the transient fusion of Httex1 to proteins (such as glutathione-S-transferase (GST), thioredoxin (TRX) or maltose-binding-protein (MBP) and/or peptides (poly-histidine) that differentially improve its expression, stability, purification and/or solubility10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28. The fusion partner is linked to Httex1 with a short sequence containing a cleavage site for proteases such as trypsin, tabacco etch virus (TEV) protease or PreScission to allow for the cleavage and release of Httex1 prior to the initiation of aggregation or purification. Shortcomings of these methods include the possibility of leaving additional residues due to non-traceless cleavage and the creation of truncated fragments due to miscleavage within the sequence of Httex1, in addition to heterogeneity due to incomplete cleavage (see Vieweg et al. for more in-depth discussion on the advantages and limitations of this approach)10. To address these limitations, we recently developed an expression strategy enabling the generation of tag-free native Httex1 for the first time by utilizing a transient N-terminal fusion of the Synechocystis sp. (Ssp) DnaB intein to Httex110. While the intein cleavage is traceless and specific and yields mg quantity of proteins, it still suffers two drawbacks that could reduce the yield: namely, premature cleavage of the intein which can occur during the expression, and the fact that cleavage occurs over several hours, which could lead to loss of protein due to aggregation, especially for Httex1 with expanded polyQ repeats.
To address these limitations and to refine our strategy for the production of native, tag-free Httex1, we developed a new expression system based on the transient fusion of SUMO, more exactly the yeast homolog Smt3 to Httex1. The application of the SUMO system for the production of recombinant proteins was first published in 200429, where an increased rate of expression and solubility of SUMO fusion protein was demonstrated. The SUMO tag can be cleaved by the ubiquitin like protein-specific protease 1 (ULP1), which does not require a recognition site, but recognizes the tertiary structure of SUMO and practically eliminates the possibility of miscleavage30. Furthermore, the ULP1-mediated cleavage is fast and traceless and does not leave additional residues behind. The premature cleavage of the fusion tag, as observed with the autocatalytic intein10, is completely avoided by the requirement of an external protease. While the SUMO strategy is nowadays widely used for recombinant protein production31,32,33, we demonstrate in this paper that it is especially useful for the generation of an intrinsically disordered, aggregation-prone, amyloidogenic protein such as Httex1. We believe that the simplicity, efficiency and robustness of our SUMO-fusion-based method will make native, tag-free Httex1 more accessible to researchers from different disciplines and eliminate the need to use non-native sequences of Httex1 in vitro. This is an important advance that will facilitate future studies to elucidate the structure-function relationship of Httex1.
The protocol describes the purification of Httex1 from 12 L of bacterial culture, but the protocol could be easily adapted for smaller or larger scale productions. The protocol describes the production of wild type Httex1 (wtHttex1) with a polyQ repeat length below (23Q) and mutant Httex1 (mHttex1) with a polyQ repeat length above (43Q) the pathogenic threshold (36Q).

<table><tbody><tr><td>Uranyl formate (UO&lt;sub&gt;2&lt;/sub&gt;(CHO2)&lt;sub&gt;2&lt;/sub&gt;)</td><td>EMS</td><td>22450</td><td></td></tr><tr><td>Formvar/carbon 200 mesh, Cu 50 grids</td><td>EMS</td><td>FCF200-Cu-50</td><td></td></tr><tr><td>High Precision Cell made of Quartz SUPRASIL 1 mm light path from Hellma Analytics</td><td>HellmaAnalytics</td><td>110-1-40</td><td></td></tr><tr><td>Buffer Substance Dulbecco&#039;s (PBS w/o Ca and Mg) ancinne ref 47302 (RT) SERVA</td><td>Witech</td><td>SVA4730203</td><td></td></tr><tr><td>Ampicillin</td><td>AxonLab</td><td>A0839.0100</td><td></td></tr><tr><td>Luria Broth (Miller&#039;s LB Broth)</td><td>&amp;nbsp;Chemie Brunschwig</td><td>1551.05</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>AxonLab</td><td>A1008.0025</td><td></td></tr><tr><td>E. coli B ER2566</td><td>NEB</td><td>NEB# E4130</td><td></td></tr><tr><td>Imidazole</td><td>Sigma</td><td>56750-500G</td><td></td></tr><tr><td>cOmplete&amp;nbsp;Protease Inhibitor Cocktail</td><td>Roche</td><td>4693116001</td><td></td></tr><tr><td>Anti-Huntingtin Antibody, a.a. 1-82</td><td>Merck Millipore Corporation</td><td>MAB5492</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG (H + L)</td><td>Licor</td><td>925-68070</td><td></td></tr><tr><td>PMSF</td><td>AxonLab</td><td>A0999.0005</td><td></td></tr><tr><td>HisPrep 16/10 column&amp;nbsp;</td><td>GE Healthcare</td><td>28936551</td><td></td></tr><tr><td>C4 HPLC column</td><td>Phenomenex</td><td>00G-4168.P0</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp; 10 &amp;micro;m C4 300 &amp;Aring;, LC Column 250 x 21.2 mm, Phenomenex, 19x10 mm guard column, not temperature jacketed</td></tr><tr><td>Acetonitrile HPLC</td><td>MachereyNagel</td><td>C2502</td><td></td></tr><tr><td>Filtre seringue Filtropur S 0,45 ul sans prefiltre sterile</td><td>Sarstedt AG</td><td>83.1826</td><td></td></tr><tr><td>Spectrophotometer semi-micro cuvette</td><td>Reactolab S.A.</td><td>2534</td><td></td></tr><tr><td>Superloop, 1/16&quot; fittings (&amp;Auml;KTAdesign), 50 ml</td><td>GE Healthcare</td><td>18111382</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>GREINER Tubes fo FPLC 16 x 100 mm, cap. 12.0 ml</td><td>Greiner Bio-One</td><td>7.160 102</td><td></td></tr><tr><td>&amp;nbsp;100 kD Microcon&amp;nbsp; fast flow filters</td><td>Merck Millipore Corporation</td><td>MRCF0R100</td><td></td></tr><tr><td>Vibra-cell VCX130 ultrasonic liquid processor</td><td>Sonics</td><td></td><td></td></tr><tr><td>&amp;Auml;kta 900 equipped with a fraction collector&amp;nbsp;</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Jasco J-815 Circular Dichroism</td><td>Jasco</td><td></td><td></td></tr><tr><td>Waters UPLC system&amp;nbsp;</td><td>Waters</td><td></td><td>C8 BEH acquity 2.1x100 mm 1.7 micron column&amp;nbsp; , preheated column (40 &amp;deg;C), flow rate of 0.6 mL/min, injection volume of 4 &amp;mu;L</td></tr><tr><td>waters HPLC system</td><td>Waters</td><td></td><td>2489 UV detector and 2535 quaternary gradient module, 20 mL loop in a FlexInject housing</td></tr><tr><td>ESI-MS: Finnigan LTQ</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>lyophylizer instrument</td><td>FreeZone 2.5 Plus</td><td></td><td></td></tr><tr><td>Tecnai Spirit BioTWIN&amp;nbsp;</td><td>FEI</td><td></td><td>electron microscope equipped with a LaB6 gun and a 4K x 4K FEI Eagle CCD camera (FEI) and operated at 80 kV</td></tr><tr><td>37 &amp;deg;C shaking&amp;nbsp; incubator</td><td>Infors HT multitron Standard</td><td></td><td></td></tr><tr><td>Biophotometer plus</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

generation of native, untagged huntingtin exon1 monomer and fibrils using a sumo fusion strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Httex1 is expressed in E. coli with an N-terminal His6-SUMO tag. The representative results of the expression and purification of the fusion protein are summarized in Figure 1. The sequence of Httex1 consists of the residues 2-90 of Htt and starts with Ala2, because Met1 is fully cleaved in vivo42. The numbering of the amino acids refers to the 23Q variant, the complete sequence of the expressed fusion protein is shown in Figure 1A. The plasmids will be deposited at Addgene in the near future to be shared with the community. A schematic of the plasmid and the expressed fusion protein is shown in Figure 1B. His6-SUMO Httex1 expresses at a medium level (Figure 1C) and most of the fusion protein is present in the soluble fraction after lysis, both for the 23Q and the 43Q variant. The fusion protein migrates higher than expected, based on the molecular weight. This is partly due to the strong fold of SUMO but mostly due to the unusual sequence composition of Httex1, containing mainly glutamine and proline residues. Both the wildtype (23Q) and the mutant (43Q) fusion protein can be enriched to ~80% purity by IMAC (Figure 1D) The presence of co-purifying host protein can be explained by the comparatively low expression level of Httex1 and the big sample volume applied to the column.
The cleavage of the His6-SUMO tag and the purification of Httex1 is shown in Figure 2A. UPLC is an efficient tool to monitor the cleavage of the His6-SUMO tag (Figure 2B). The original peak of the fusion protein is consumed and two new and well-separated peaks corresponding the His6-SUMO tag and Httex1 appear. The cleavage reaction is finished in 10-20 min. The Western Blot (WB) is too slow to monitor the cleavage reaction efficiently, but it has been included in the figure for reference and to demonstrate the completeness of the SUMO cleavage. Both Httex1 23Q and 43Q can be separated well from the His6-SUMO tag by RP-HPLC (Figure 2C) and were obtained in high purity as shown by UPLC, MS and SDS-PAGE analysis (Figure 2D).
To illustrate that the Httex1 proteins prepared by this method retain the expected aggregation properties of Httex1, we assessed the fibrillization kinetics of mutant Httex1 at 37 &#176;C by a sedimentation assay, monitored the changes in secondary structure by CD spectroscopy, and characterized the morphology of the aggregates by TEM. A representative data set of the aggregation kinetics of mHttex1 fibril formation as determined by a sedimentation assay is shown inFigure 3A. The loss of soluble Httex1 43Q over time, due to fibril formation was quantified by UPLC. We observe a complete depletion of soluble protein after 48 hours of incubation. Additionally, we determined the secondary structure of the protein by CD spectroscopy (Figure 3B). Httex1 43Q shifts from unstructured (&#955;min 205 nm) to mainly &#946;-sheet rich conformation (&#955;min 215 nm) after 48 hours of incubation. This structural change is accompanied by the formation of long fibrillar aggregates as observable by TEM at 48 hours (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57506/57506fig1.jpg" /> 
Figure 1. Expression and purification of the His6-SUMO Httex1 fusion protein.&#160; 
(A) The amino acid sequence of the His6-SUMO-Httex1-QN fusion constructs (His6-SUMO in green and Httex1-QN in orange); (B) Schematic overview of the expression and purification of the fusion protein; (C) SDS-PAGE analysis of the expression and the solubility of the fusion protein after lysis; (D) Chromatogram of the IMAC purification of the fusion protein and analysis of the fractions by SDS-PAGE (red bar: unbound fraction, blue bar: wash fraction, black bar: fractions containing the elution peak);&#160;<a href="/files/ftp_upload/57506/57506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57506/57506fig2.jpg" /> 
Figure 2. Cleavage of the His6 SUMO tag and purification of tag-free Httex1-QN proteins. 
(A) schematic overview; (B) analysis of the cleavage of the SUMO tag with ULP1 by UPLC (blue: before addition of ULP1; black: 20 min (23Q), respectively 10 min (43Q) after addition of ULP1) and WB (MAB5492 1:2000, secondary goat anti mouse antibody 1:5000); (C) Chromatogram of the preparative RP-HPLC purification of Httex1; D: analysis of the purified Httex1 by UPLC, SDS-PAGE and ESI-MS; the expected molecular weight is 9943 Da (23Q) and 12506 Da (43Q) respectively.&#160;<a href="/files/ftp_upload/57506/57506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57506/57506fig3.jpg" /> 
Figure 3. Aggregation of Httex1-43Q: (A) Sedimentation assay based on UPLC. (B) CD spectra of the secondary structure at 0 h and 48 h. (C) TEM micrographs of the aggregates at 48 h (scale bars are 200 nm). <a href="/files/ftp_upload/57506/57506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy at JoVE.com

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

generation-native-untagged-huntingtin-exon1-monomer-fibrils-using

Research

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Biochemistry

7.9K Views.  Ecole Polytechnique Fédérale de Lausanne (EPFL). Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Video: Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetics

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

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.

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 ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Neuroscience

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Medicine

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biology

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

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

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Rapid Generation of Amyloid from Native Proteins In vitro

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast