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/pubmed?term=((Huntington%27s+disease%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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/pubmed?term=((Huntington%27s+disease%3A+a+clinical+review%5BTitle%5D)+AND+%22European+Journal+of+Neurology%22%5BJournal%5D)">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/pubmed?term=((Trinucleotide+repeat+length+instability+and+age+of+onset+in+Huntington%27s+disease%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((A+novel+gene+containing+a+trinucleotide+repeat+that+is+expanded+and+unstable+on+Huntington%27s+disease+chromosomes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Targeted+disruption+of+the+Huntington%27s+disease+gene+results+in+embryonic+lethality+and+behavioral+and+morphological+changes+in+heterozygotes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Inactivation+of+Hdh+in+the+brain+and+testis+results+in+progressive+neurodegeneration+and+sterility+in+mice%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">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/pubmed?term=((Phosphorylation+of+huntingtin+by+cyclin-dependent+kinase+5+is+induced+by+DNA+damage+and+regulates+wild-type+and+mutant+huntingtin+toxicity+in+neurons%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Elimination+of+huntingtin+in+the+adult+mouse+leads+to+progressive+behavioral+deficits%2C+bilateral+thalamic+calcification%2C+and+altered+brain+iron+homeostasis%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+modulates+transcription%2C+occupies+gene+promoters+in+vivo%2C+and+binds+directly+to+DNA+in+a+polyglutamine-dependent+manner%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((The+biology+of+huntingtin%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Formation+of+neuronal+intranuclear+inclusions+underlies+the+neurological+dysfunction+in+mice+transgenic+for+the+HD+mutation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Aggregation+of+huntingtin+in+neuronal+intranuclear+inclusions+and+dystrophic+neurites+in+brain%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Nuclear+and+neuropil+aggregates+in+Huntington%27s+disease%3A+Relationship+to+neuropathology%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((A+YAC+mouse+model+for+Huntington%27s+disease+with+full-length+mutant+huntingtin%2C+cytoplasmic+toxicity%2C+and+selective+striatal+neurodegeneration%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Polyglutamine+aggregation+in+Huntington+disease%3A+does+structure+determine+toxicity%5BTitle%5D)+AND+%22Molecular+Neurobiology%22%5BJournal%5D)">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/pubmed?term=((The+neuropathology+of+Huntington%27s+disease%5BTitle%5D)+AND+%22Current+Topics+in+Behavioral+Neurosciences%22%5BJournal%5D)">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/pubmed?term=((Beta+conformation+of+polyglutamine+track+revealed+by+a+crystal+structure+of+huntingtin+N-terminal+region+with+insertion+of+three+histidine+residues%5BTitle%5D)+AND+%22Prion%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+exon+1+fibrils+feature+an+interdigitated+%CE%B2-hairpin-based+polyglutamine+core%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Huntingtin exon 1 fibrils feature an interdigitated β-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/pubmed?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%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+facilitates+polycomb+repressive+complex+2%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">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/pubmed?term=((Expression+and+characterization+of+full-length+human+huntingtin%2C+an+elongated+HEAT+repeat+protein%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Huntingtin%27s+spherical+solenoid+structure+enables+polyglutamine+tract-dependent+modulation+of+its+structure+and+function%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">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/pubmed?term=((Scalable+production+in+human+cells+and+biochemical+characterization+of+full-length+normal+and+mutant+huntingtin%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">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/pubmed?term=((The+cryo-electron+microscopy+structure+of+huntingtin%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Design+and+characterization+of+mutant+and+wildtype+huntingtin+proteins+produced+from+a+toolkit+of+scalable+eukaryotic+expression+systems%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((HAP40+orchestrates+huntingtin+structure+for+1+differential+interaction+with+polyglutamine+2+expanded+exon+1%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">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/pubmed?term=((Pathological+polyQ+expansion+does+not+alter+the+conformation+of+the+Huntingtin-HAP40+complex%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+phosphorylation+acts+as+a+molecular+switch+for+anterograde%2Fretrograde+transport+in+neurons%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">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/pubmed?term=((IKK+phosphorylates+Huntingtin+and+targets+it+for+degradation+by+the+proteasome+and+lysosome%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Post-translational+modifications+%28PTMs%29%2C+identified+on+endogenous+Huntingtin%2C+cluster+within+proteolytic+domains+between+HEAT+repeats%5BTitle%5D)+AND+%22Journal+of+Proteome+Research%22%5BJournal%5D)">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/pubmed?term=((A+defined+methodology+for+reliable+quantification+of+western+blot+data%5BTitle%5D)+AND+%22Molecular+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Combination+of+SEC%2FMALS+experimental+procedures+and+theoretical+analysis+for+studying+the+solution+properties+of+macromolecules%5BTitle%5D)+AND+%22Journal+of+Biochemical+and+Biophysical+Methods%22%5BJournal%5D)">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/pubmed?term=((Oligomeric+states+of+proteins+determined+by+size-exclusion+chromatography+coupled+with+light+scattering%2C+absorbance%2C+and+refractive+index+detectors%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors.</a> Methods in Molecular Biology. 328, Clifton, N.J. 97-112 (2006).</li>
<li>McMeekin, T. L., Wilensky, M., Groves, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Refractive+indices+of+proteins+in+relation+to+amino+acid+composition+and+specific+volume%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+has+a+membrane+association+signal+that+can+modulate+huntingtin+aggregation%2C+nuclear+entry+and+toxicity%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">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/pubmed?term=((Huntingtin+interactions+with+membrane+phospholipids%3A+Strategic+targets+for+therapeutic+intervention%5BTitle%5D)+AND+%22Journal+of+Huntington%27s+Disease%22%5BJournal%5D)">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/pubmed?term=((Membrane+interactions+of+the+amphipathic+amino+terminus+of+huntingtin%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Membrane interactions of the amphipathic amino terminus of huntingtin.</a> Biochemistry. 52 (5), 847-858 (2013).</li>
<li>Wittig, I., Braun, H. P., Schägger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blue+native+PAGE%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Altered+co-translational+processing+plays+a+role+in+huntington%27s+pathogenesis-A+hypothesis%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Proliferation+control+strategies+to+improve+productivity+and+survival+during+CHO+based+production+culture%3A+A+summary+of+recent+methods+employed+and+the+effects+of+proliferation+control+in+product+secreting+CHO+cell+lines%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">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><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 &amp;micro;m x 4.6 mm x 150 mm&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Protocol Section Number-1.2</td></tr><tr><td>Glycerol</td><td>Fisher Scientific Glycerol (Certified ACS)</td><td>G33-4</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&lt;br/&gt; 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&lt;br/&gt; Protocol Section Number-2.2</td></tr><tr><td>NuPAGE 3&amp;ndash;8% Tris acetate gels</td><td>Invitrogen</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

2.5K 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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

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, ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Neuroscience

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...