Protein transduction: Delivery of tat-GTPase fusion proteins into mammalian cells

Abstract

Publisher Summary This chapter describes the methodology to generate and transduce full-length Tat fusion proteins that can be applied to a broad spectrum of proteins independent of size or function. Bacterially expressed N'-terminal in-frame Tat fusion proteins are isolated from bacteria by sonication in 8 M urea. The use of 8 M urea achieves two goals. First, the majority of recombinant proteins in bacteria are present in inclusion bodies as denatured insoluble proteins, especially full-length proteins. Sonication in urea solubilizes this material, thus allowing for its isolation. Second, denatured Tat fusion proteins have an enhanced potential to elicit biological responses. The denatured Tat fusion proteins are made soluble in an aqueous buffer and added directly to the medium of cells in tissue culture. To date, this strategy is used to generate and transduce more than 60 full-length proteins and domains from 15 to 120 kDa from a variety of classes, suggesting that many proteins may be transduced into cells. Transduction of full-length Tat fusion proteins directly into ∼100% of primary or transformed cells has broad implications for manipulating intracellular processes in both experimental in vitro tissue culture systems and animal models.