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.

<ol>
	<li>Walker, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease.">Huntington's disease.</a> Lancet. 369 (9557), 218-228 (2007).</li><li>McColgan, P., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+a+clinical+review.">Huntington's disease: a clinical review.</a> European Journal of Neurology. 25 (1), 24-34 (2018).</li><li>Duyao, M., 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=Trinucleotide+repeat+length+instability+and+age+of+onset+in+Huntington's+disease.">Trinucleotide repeat length instability and age of onset in Huntington's disease.</a> Nature Genetics. 4 (4), 387-392 (1993).</li><li>MacDonald, M. E., 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+novel+gene+containing+a+trinucleotide+repeat+that+is+expanded+and+unstable+on+Huntington's+disease+chromosomes.">A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.</a> Cell. 72 (6), 971-983 (1993).</li><li>Nasir, J., 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=Targeted+disruption+of+the+Huntington's+disease+gene+results+in+embryonic+lethality+and+behavioral+and+morphological+changes+in+heterozygotes.">Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes.</a> Cell. 81 (5), 811-823 (1995).</li><li>Dragatsis, I., Levine, M. S., Zeitlin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Hdh+in+the+brain+and+testis+results+in+progressive+neurodegeneration+and+sterility+in+mice.">Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice.</a> Nature Genetics. 26 (3), 300-306 (2000).</li><li>Anne, S. L., Saudou, F., Humbert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+huntingtin+by+cyclin-dependent+kinase+5+is+induced+by+DNA+damage+and+regulates+wild-type+and+mutant+huntingtin+toxicity+in+neurons.">Phosphorylation of huntingtin by cyclin-dependent kinase 5 is induced by DNA damage and regulates wild-type and mutant huntingtin toxicity in neurons.</a> Journal of Neuroscience. 27 (27), 7318-7328 (2007).</li><li>Dietrich, P., Johnson, I. M., Alli, S., Dragatsis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+huntingtin+in+the+adult+mouse+leads+to+progressive+behavioral+deficits,+bilateral+thalamic+calcification,+and+altered+brain+iron+homeostasis.">Elimination of huntingtin in the adult mouse leads to progressive behavioral deficits, bilateral thalamic calcification, and altered brain iron homeostasis.</a> PLoS Genetics. 13 (7), 1006846 (2017).</li><li>Dragatsis, I., 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=Effect+of+early+embryonic+deletion+of+huntingtin+from+pyramidal+neurons+on+the+development+and+long-term+survival+of+neurons+in+cerebral+cortex+and+striatum.">Effect of early embryonic deletion of huntingtin from pyramidal neurons on the development and long-term survival of neurons in cerebral cortex and striatum.</a> Neurobiology of Disease. 111, 102-117 (2018).</li><li>Benn, C. L., 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=Huntingtin+modulates+transcription,+occupies+gene+promoters+in+vivo,+and+binds+directly+to+DNA+in+a+polyglutamine-dependent+manner.">Huntingtin modulates transcription, occupies gene promoters in vivo, and binds directly to DNA in a polyglutamine-dependent manner.</a> Journal of Neuroscience. 28 (42), 10720-10733 (2008).</li><li>Saudou, F., Humbert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+huntingtin.">The biology of huntingtin.</a> Neuron. 89 (5), 910-926 (2016).</li><li>Davies, S. W., 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=Formation+of+neuronal+intranuclear+inclusions+underlies+the+neurological+dysfunction+in+mice+transgenic+for+the+HD+mutation.">Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation.</a> Cell. 90 (3), 537-548 (1997).</li><li>DiFiglia, M., 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=Aggregation+of+huntingtin+in+neuronal+intranuclear+inclusions+and+dystrophic+neurites+in+brain.">Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.</a> Science. 277 (5334), 1990-1993 (1997).</li><li>Gutekunst, C. A., 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=Nuclear+and+neuropil+aggregates+in+Huntington's+disease:+Relationship+to+neuropathology.">Nuclear and neuropil aggregates in Huntington's disease: Relationship to neuropathology.</a> Journal of Neuroscience. 19 (7), 2522-2534 (1999).</li><li>Hodgson, J. G., 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+YAC+mouse+model+for+Huntington's+disease+with+full-length+mutant+huntingtin,+cytoplasmic+toxicity,+and+selective+striatal+neurodegeneration.">A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.</a> Neuron. 23 (1), 181-192 (1999).</li><li>Hoffner, G., Djian, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyglutamine+aggregation+in+Huntington+disease:+does+structure+determine+toxicity.">Polyglutamine aggregation in Huntington disease: does structure determine toxicity.</a> Molecular Neurobiology. 52 (3), 1297-1314 (2015).</li><li>Waldvogel, H. J., Kim, E. H., Tippett, L. J., Vonsattel, J. P. G., Faull, R. L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropathology+of+Huntington's+disease.">The neuropathology of Huntington's disease.</a> Current Topics in Behavioral Neurosciences. 22, 33-80 (2014).</li><li>Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+conformation+of+polyglutamine+track+revealed+by+a+crystal+structure+of+huntingtin+N-terminal+region+with+insertion+of+three+histidine+residues.">Beta conformation of polyglutamine track revealed by a crystal structure of huntingtin N-terminal region with insertion of three histidine residues.</a> Prion. 7 (3), 221-228 (2013).</li><li>Hoop, C. L., 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=Huntingtin+exon+1+fibrils+feature+an+interdigitated+&#946;-hairpin-based+polyglutamine+core.">Huntingtin exon 1 fibrils feature an interdigitated &#946;-hairpin-based polyglutamine core.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (6), 1546-1551 (2016).</li><li>Vieweg, S., Ansaloni, A., Wang, Z. M., Warner, J. B., Lashuel, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intein-based+strategy+for+the+production+of+tag-free+huntingtin+exon+1+proteins+enables+new+insights+into+the+polyglutamine+dependence+of+Httex1+aggregation+and+fibril+formation.">An intein-based strategy for the production of tag-free huntingtin exon 1 proteins enables new insights into the polyglutamine dependence of Httex1 aggregation and fibril formation.</a> Journal of Biological Chemistry. 291 (23), 12074-12086 (2016).</li><li>Seong, I. S., 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=Huntingtin+facilitates+polycomb+repressive+complex+2.">Huntingtin facilitates polycomb repressive complex 2.</a> Human Molecular Genetics. 19 (4), 573-583 (2009).</li><li>Li, W., Serpell, L. C., Carter, W. J., Rubinsztein, D. C., Huntington, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+full-length+human+huntingtin,+an+elongated+HEAT+repeat+protein.">Expression and characterization of full-length human huntingtin, an elongated HEAT repeat protein.</a> Journal of Biological Chemistry. 281 (23), 15916-15922 (2006).</li><li>Vijayvargia, R., 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=Huntingtin's+spherical+solenoid+structure+enables+polyglutamine+tract-dependent+modulation+of+its+structure+and+function.">Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function.</a> eLife. 5, 11184 (2016).</li><li>Huang, B., 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=Scalable+production+in+human+cells+and+biochemical+characterization+of+full-length+normal+and+mutant+huntingtin.">Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.</a> PLoS ONE. 10 (3), 0121055 (2015).</li><li>Guo, Q., 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=The+cryo-electron+microscopy+structure+of+huntingtin.">The cryo-electron microscopy structure of huntingtin.</a> Nature. 555 (7694), 117-120 (2018).</li><li>Harding, R. J., 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=Design+and+characterization+of+mutant+and+wildtype+huntingtin+proteins+produced+from+a+toolkit+of+scalable+eukaryotic+expression+systems.">Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems.</a> Journal of Biological Chemistry. 294 (17), 6986-7001 (2019).</li><li>Harding, R. J., 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=HAP40+orchestrates+huntingtin+structure+for+1+differential+interaction+with+polyglutamine+2+expanded+exon+1.">HAP40 orchestrates huntingtin structure for 1 differential interaction with polyglutamine 2 expanded exon 1.</a> bioRxiv. , (2021).</li><li>Huang, B., 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=Pathological+polyQ+expansion+does+not+alter+the+conformation+of+the+Huntingtin-HAP40+complex.">Pathological polyQ expansion does not alter the conformation of the Huntingtin-HAP40 complex.</a> Structure. 29 (8), 804-809 (2021).</li><li>Colin, E., 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=Huntingtin+phosphorylation+acts+as+a+molecular+switch+for+anterograde/retrograde+transport+in+neurons.">Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons.</a> EMBO Journal. 27 (15), 2124-2134 (2008).</li><li>Thompson, L. M., 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=IKK+phosphorylates+Huntingtin+and+targets+it+for+degradation+by+the+proteasome+and+lysosome.">IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome.</a> Journal of Cell Biology. 187 (7), 1083-1099 (2009).</li><li>Ratovitski, T., 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=Post-translational+modifications+(PTMs),+identified+on+endogenous+Huntingtin,+cluster+within+proteolytic+domains+between+HEAT+repeats.">Post-translational modifications (PTMs), identified on endogenous Huntingtin, cluster within proteolytic domains between HEAT repeats.</a> Journal of Proteome Research. 16 (8), 2692-2708 (2017).</li><li>Taylor, S. C., Berkelman, T., Yadav, G., Hammond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+methodology+for+reliable+quantification+of+western+blot+data.">A defined methodology for reliable quantification of western blot data.</a> Molecular Biotechnology. 55 (3), 217-226 (2013).</li><li>Tarazona, M. P., Saiz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+SEC/MALS+experimental+procedures+and+theoretical+analysis+for+studying+the+solution+properties+of+macromolecules.">Combination of SEC/MALS experimental procedures and theoretical analysis for studying the solution properties of macromolecules.</a> Journal of Biochemical and Biophysical Methods. 56 (1-3), 95-116 (2003).</li><li>Folta-Stogniew, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomeric+states+of+proteins+determined+by+size-exclusion+chromatography+coupled+with+light+scattering,+absorbance,+and+refractive+index+detectors.">Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors.</a> Methods in Molecular Biology. 328, 97-112 (2006).</li><li>McMeekin, T. L., Wilensky, M., Groves, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refractive+indices+of+proteins+in+relation+to+amino+acid+composition+and+specific+volume.">Refractive indices of proteins in relation to amino acid composition and specific volume.</a> Biochemical and Biophysical Research Communications. 7 (2), 151-156 (1962).</li><li>Atwal, R. S., 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=Huntingtin+has+a+membrane+association+signal+that+can+modulate+huntingtin+aggregation,+nuclear+entry+and+toxicity.">Huntingtin has a membrane association signal that can modulate huntingtin aggregation, nuclear entry and toxicity.</a> Human Molecular Genetics. 16 (21), 2600-2615 (2007).</li><li>Kegel-Gleason, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntingtin+interactions+with+membrane+phospholipids:+Strategic+targets+for+therapeutic+intervention.">Huntingtin interactions with membrane phospholipids: Strategic targets for therapeutic intervention.</a> Journal of Huntington's Disease. 2 (3), 239-250 (2013).</li><li>Michalek, M., Salnikov, E. S., Werten, S., Bechinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+interactions+of+the+amphipathic+amino+terminus+of+huntingtin.">Membrane interactions of the amphipathic amino terminus of huntingtin.</a> Biochemistry. 52 (5), 847-858 (2013).</li><li>Wittig, I., Braun, H. P., Sch&#228;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nature Protocols. 1 (1), 418-428 (2006).</li><li>Nissley, D. A., O'Brien, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+co-translational+processing+plays+a+role+in+huntington's+pathogenesis-A+hypothesis.">Altered co-translational processing plays a role in huntington's pathogenesis-A hypothesis.</a> Frontiers in Molecular Neuroscience. 9, 54 (2016).</li><li>Kumar, N., Gammell, P., Clynes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+control+strategies+to+improve+productivity+and+survival+during+CHO+based+production+culture:+A+summary+of+recent+methods+employed+and+the+effects+of+proliferation+control+in+product+secreting+CHO+cell+lines.">Proliferation control strategies to improve productivity and survival during CHO based production culture: A summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines.</a> Cytotechnology. 53 (1-3), 33-46 (2007).</li></ol>

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 ...

efficient-scalable-production-full-length-human-huntingtin-variants

Research

JoVE Journal

Biochemistry

2.4K Views.  Curia Global Inc.. We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

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

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

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

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

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&amp;#945;

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

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 ...

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.

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, ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...