A rapid protein expression and purification system using Chinese hamster ovary cells expressing retrovirus receptor.
Conventional stable protein expression systems using mammalian cells include a time-consuming step of antibiotic resistance-based cell cloning. Here, we report a rapid flow cytometry-based method for the collection of retrovirus vector-infected Chinese hamster ovary (CHO) cells that express desired proteins. The vector carries the genes for green fluorescent protein (GFP), as a marker, and glutathione-S-transferase (GST), to express the desired protein as a GST-fusion construct. To render CHO cells susceptible to retrovirus infection, they were forced to express EcoR, the receptor for retroviruses. After infection, cells expressing desired proteins were collected by flow cytometry as a GFP-positive population, and the desired proteins were purified by glutathione affinity chromatography. This method reduces the time required between infection of cells and purification of a desired protein from several months to approximately 2 weeks.
Suzuki
Jun
J
Laboratory of Molecular Oncology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Fukuda
Machi
M
Kawata
Shigehisa
S
Maruoka
Masahiro
M
Kubo
Yoko
Y
Takeya
Tatsuo
T
Shishido
Tomoyuki
T
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D005434:Flow Cytometry; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors
D005819:Genetic Markers; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase