Production of cell lines secreting TAT fusion proteins.
Transduction of proteins and other macromolecules constitutes a potent technology to analyze cell functions and to achieve therapeutic interventions. In general, fusion proteins with protein transduction domains, such as TAT, are produced in a bacterial expression system. Here we describe the generation of a mammalian expression vector coding for TAT-EGFP fusion protein. Transfection of CHO-K1 cells by this vector and subsequent selection by Zeocin resulted in cell lines that express and secrete EGFP, a variant of the green fluorescent protein GFP. The ultimate cell line was produced by first cloning the stable integrants and subsequent selection of EGFP-expressing cells by flow cytometric sorting. In the resulting cell line approximately 98% of cells express EGFP. Using the same methodology, we generated cell lines that express DsRed fluorescent protein. The advantages of using such a mammalian expression system include the ease of generating TAT fusion proteins and the potential for sustained production of such proteins in vitro and, potentially, in vivo.
Barka
Tibor
T
Center for Anatomy and Functional Morphology and Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, USA. Tibor.Barka@mssm.edu
Gresik
Edward S
ES
Henderson
Scott C
SC
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D002470:Cell Survival; D006224:Cricetinae; D005240:Feasibility Studies; D005434:Flow Cytometry; D015696:Gene Products, tat / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D000097573:Red Fluorescent Protein
D015696:Gene Products, tat; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein