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 (>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.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2021 |
|---|---|
| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2021 |
|---|---|
| Enthalten in: |
Journal of visualized experiments : JoVE - (2021), 178 vom: 10. Dez. |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Pace, James B [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
|---|
| Anmerkungen: |
Date Completed 05.04.2022 Date Revised 05.04.2022 published: Electronic Citation Status MEDLINE |
|---|
| doi: |
10.3791/63190 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM334939623 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM334939623 | ||
| 003 | DE-627 | ||
| 005 | 20231225224749.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231225s2021 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.3791/63190 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1116.xml |
| 035 | |a (DE-627)NLM334939623 | ||
| 035 | |a (NLM)34958078 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Pace, James B |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System |
| 264 | 1 | |c 2021 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 05.04.2022 | ||
| 500 | |a Date Revised 05.04.2022 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 (>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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Video-Audio Media | |
| 650 | 7 | |a Huntingtin Protein |2 NLM | |
| 700 | 1 | |a Huang, Nian N |e verfasserin |4 aut | |
| 700 | 1 | |a Séguin, Jacqueline P |e verfasserin |4 aut | |
| 700 | 1 | |a Esquina, Candi |e verfasserin |4 aut | |
| 700 | 1 | |a Olin, Erin |e verfasserin |4 aut | |
| 700 | 1 | |a Zhu, Guangyu |e verfasserin |4 aut | |
| 700 | 1 | |a Carr, Grant |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Journal of visualized experiments : JoVE |d 2006 |g (2021), 178 vom: 10. Dez. |w (DE-627)NLM181595761 |x 1940-087X |7 nnns |
| 773 | 1 | 8 | |g year:2021 |g number:178 |g day:10 |g month:12 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.3791/63190 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |j 2021 |e 178 |b 10 |c 12 | ||