Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO L...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3235087?pdf=render |
| _version_ | 1811191620596400128 |
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| author | Sonja Wilke Lothar Groebe Vitali Maffenbeier Volker Jäger Manfred Gossen Jörn Josewski Agathe Duda Lilia Polle Raymond J Owens Dagmar Wirth Dirk W Heinz Joop van den Heuvel Konrad Büssow |
| author_facet | Sonja Wilke Lothar Groebe Vitali Maffenbeier Volker Jäger Manfred Gossen Jörn Josewski Agathe Duda Lilia Polle Raymond J Owens Dagmar Wirth Dirk W Heinz Joop van den Heuvel Konrad Büssow |
| author_sort | Sonja Wilke |
| collection | DOAJ |
| description | Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP). |
| first_indexed | 2024-04-11T23:38:53Z |
| format | Article |
| id | doaj.art-70ab6f71a887448c809ae9610d88d32e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-11T23:38:53Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-70ab6f71a887448c809ae9610d88d32e2022-12-22T03:56:50ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-01612e2782910.1371/journal.pone.0027829Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.Sonja WilkeLothar GroebeVitali MaffenbeierVolker JägerManfred GossenJörn JosewskiAgathe DudaLilia PolleRaymond J OwensDagmar WirthDirk W HeinzJoop van den HeuvelKonrad BüssowStudying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).http://europepmc.org/articles/PMC3235087?pdf=render |
| spellingShingle | Sonja Wilke Lothar Groebe Vitali Maffenbeier Volker Jäger Manfred Gossen Jörn Josewski Agathe Duda Lilia Polle Raymond J Owens Dagmar Wirth Dirk W Heinz Joop van den Heuvel Konrad Büssow Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. PLoS ONE |
| title | Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. |
| title_full | Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. |
| title_fullStr | Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. |
| title_full_unstemmed | Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. |
| title_short | Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. |
| title_sort | streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development |
| url | http://europepmc.org/articles/PMC3235087?pdf=render |
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