A single plasmid transfection that offers a significant advantage associated with puromycin selection in Drosophila Schneider S2 cells expressing heterologous proteins

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.

Fusion of fluorescent protein to puromycin N-acetyltransferase is useful in Drosophila Schneider S2 cells expressing heterologous proteins

“…Using pMT-PURO we have successfully expressed several human and murine proteins related to coagulation and fibrinolysis, e.g., plasminogen, urokinasetype plasminogen activator, coagulation factor XII, high molecular weight kininogen, and prekallikrein along with a large number of variants of these proteins (Iwaki and Castellino 2008). The previous method was well established to synthesize large amounts of recombinant proteins.…”

“…After establishing the co-expressing vector, we also generated an all-in-one single plasmid pMT-PURO, which enables to express gene of interests as well as a selection gene ''pac'' (Iwaki and Castellino 2008).…”

A single plasmid transfection that offers a significant advantage associated with puromycin selection, fluorescence-assisted cell sorting, and doxycycline-inducible protein expression in mammalian cells

“…The cDNAs encoding for rtTA, PURO, and DsRED2 were digested from the already subcloned plasmids pTet-On-Advanced, pCoPURO (Iwaki and Castellino 2008;Iwaki et al 2003), and pDsRED2 (Takara Bio), respectively, with the restriction enzymes EcoRI and NotI (Fig. 1c).…”

Dpy-19 like 3-mediated C-mannosylation and expression levels of RPE-spondin in human tumor cell lines

“…The reaction was conducted in a C1000™ thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the cycling conditions were as follows: 30 cycles at 98˚C for 10 sec, 55˚C for 5 sec and 72˚C for 5 sec. Subsequently, the RPESP-Myc-His 6 cDNA was subcloned into the BglII/MluI restriction sites of pMT-PURO (RIKEN BioResource Center) (20), resulting in the expression of RPESP-Myc-His 6 protein. Human DPY19L1, DPY19L2, DPY19L3 and DPY19L4 cDNAs, which were subcloned into pIZ/V5-his vectors (Thermo Fisher Scientific, Inc.), were constructed previously (12).…”