Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death.
Currently, delivery of expression vectors, proteins, and/or pharmacologically important peptidyl mimetics to target cells is problematic because of the low percentage of cells targeted, overexpression, size constraints, and bioavailability. Concentration-dependent transduction of full-length proteins and domains directly into cells would serve to alleviate these problems. Previous researchers have demonstrated the ability of proteins linked to the human immunodeficiency virus (HIV) Tat transduction domain to transduce into cells; but because of inefficiencies, this methodological potential has not significantly progressed since 1988. We describe, in this chapter, a significant increase in transduction efficiency of proteins and ease of use by (1) generation of a Tat protein transduction domain in-frame bacterial expression vector, pTAT-HA, and (2) development of a purification protocol yielding denatured proteins. We have transduced full-length Tat fusion proteins ranging in size from 15 to 115 kDa into approximately 100% of all target cells examined, including peripheral blood lymphocytes, all cells present in whole blood, bone marrow stem cells, diploid fibroblasts, fibrosarcoma cells, and keratinocytes. Transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 10 min. We conclude that our methodology generates highly efficient transducible proteins that are biologically active and have broad potential in the manipulation of biological experimental systems, such as apoptotic induction, cell cycle progression, and differentiation, and in the delivery of pharmacologically relevant proteins.
Howard Hughes Medical Institute, St. Louis, Missouri, USA.
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Vocero-Akbani
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NA
Lissy
S F
SF
Dowdy
D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D015696:Gene Products, tat / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D015696:Gene Products, tat; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins