Abstract
The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively. This system can establish both transient and stable transformants with various selection markers. The generation of stable cell lines for increased expression or large scale expression of the desired protein is currently accomplished by cotransfection of both the expression and selection vectors. The selection vectors, pCoHYGRO and pCoBLAST, are commercially available using hygromycin-B and blasticidin S, respectively. Recently, we generated a plasmid, pCoPURO, for selection of transfected S2 cells using puromycin, which allows significant acceleration of the selection time. Although co-transfection of the selection marker with the plasmid for heterologous protein expression is functional in stable expression at short culture periods, the expression levels of stable transformants are continuously decreased during long culture times. To overcome this limitation, we generated pMT-PURO, a new plasmid that contains both the expression cassette and puromycin selection marker in a single plasmid. This system allows rapid selection and maintenance of the transformed S2 lines for extended culture periods.
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Acknowledgments
We thank Mr. Liang Zhong for inserting the puromycin cassette into pMT/BiP/V5-HisA, Ms. Mariana Figuera Losada for expressing human plasminogen and its variants, Ms. Diana Cruz-Topete for expressing human and murine plasminogen and their variants, and Dr. Qihua Fu for expressing human and murine urokinase-type plasminogen activator and their variants with this system. We also thank many laboratories who used pCoPURO and provided to us important feedback. This work was supported in part by grants HL13423 and HL19982 (to FJC).
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Iwaki, T., Castellino, F.J. A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins. Cytotechnology 57, 45–49 (2008). https://doi.org/10.1007/s10616-008-9129-0
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DOI: https://doi.org/10.1007/s10616-008-9129-0