Elsevier

Methods in Enzymology

Volume 322, 2000, Pages 508-521
Methods in Enzymology

[44] - Transduction of Full-Length Tat Fusion Proteins Directly into Mammalian Cells: Analysis of T Cell Receptor Activation-Induced Cell Death

https://doi.org/10.1016/S0076-6879(00)22046-6Get rights and content

Introduction

Deletion of antigen-activated T cells after an immune response and during peripheral negative selection after strong T cell receptor (TCR) engagement of cycling T cells occurs by an apoptotic process termed TCR activation-induced cell death (TCR-AID).1 Execution of TCR-AID requires both the presence of the stimulated T cell at a late G1-phase cell cycle position, termed the death check point, and functional retinoblastoma protein (pRb).2 To dissect the question of specific cell cycle sensitivity to AID required the ability to manipulate the biology and in vivo biochemistry of ∼100% of T cells in a population.

Transfection or viral introduction of cDNA expression vectors and microinjection of proteins into cells present various difficulties, including massive overexpression, broad cell-to-cell intracellular concentration ranges of expressed proteins, and a low percentage of cells targeted.3 , 4 In addition, the use of antisense approaches to manipulate intracellular processes have both specific gene and cell type restrictions. Thus, the ability to manipulate cellular processes by introduction of full-length proteins or protein domains in a concentration-dependent fashion into 100% of cells would serve to alleviate these technological problems.

In 1988, Green and Loewenstein5 and Frankel and Pabo6 independently uncovered the ability of HIV Tat protein to cross cell membranes. In 1994, Fawell et al.7 expanded on this observation by demonstrating that heterologous proteins chemically cross-linked to a 36-amino acid domain of Tat were able to transduce into cells. However, these reports did not develop into a broadly usable method to efficiently transduce proteins into cells. Subsequent to the Tat discovery, other transduction domains have been identified that reside in the Antennapedia μlntp) protein from Drosophila8 and herpes simplex virus (HSV) VP22 protein.9 The exact mechanism of transduction across cellular membranes remains unclear; however, small Tat peptides have been shown to transduce into cells at 4° in a receptorless fashion.10 This observation suggests that all cell types are potentially targetable by this methodology.

Although Tat–mediated protein transduction was first discovered in 1988, no method to harness this technological potential has been devised. We describe the development of full-length protein transduction methodology by utilization of urea-denatured, shock-misfolded, genetic in-frame Tat fusion proteins that can be applied to a broad spectrum of proteins regardless of size or function. Briefly, 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. Sonication in urea solubilizes this material, thus allowing for its isolation. Second, transduction of denatured Tat fusion proteins elicits biological responses more efficiently than correctly folded soluble proteins.11 The denatured proteins are made aqueously soluble and added directly to the tissue culture medium. We have transduced Tat fusion proteins into a variety of primary and transformed cell types, including peripheral blood lymphocytes (PBLs), diploid human fibroblasts, keratinocytes, bone marrow stem cells, osteoclasts, fibrosarcoma cells, leukemic T cells, osteosarcoma, glioma, hepatocellular carcinoma, renal carcinoma, NIH 3T3 cells, and all cells present in whole blood, including both nucleated and enucleated cells.2, 11, 12, 13 Most importantly, we have generated and transduced more than 50 full-length proteins and domains from 15 to 115 kDa by this method, suggesting that most proteins may be transduced into cells.11

Section snippets

Buffers

  • Buffer Z: 8 M urea–100 mM NaCl–20 mM HEPES (pH 8.0)

  • Buffer A: 50 mM NaCl–20 mM HEPES (pH 8.0)

  • Buffer B: 1 M NaCl-20 mM HEPES (pH 8.0)

  • Phosphate-buffered saline (PBS)

  • Paraformaldehyde (4%, w/v)

  • Imidazole (5 M)

Reagents

  • pTat–HA plasmid

  • BL21(DE3)LysS bacteria (Novagen, Madison, WI)

  • 12CA5 anti-hemagglutinin (HA) antibodies (BabCO, Berkeley, CA)

  • Protein fast protein liquid chromatography (FITC) labeling kit (Pierce, Rockford, IL)

  • Ni-NTA resin (Qiagen, Chatsworth, CA)

  • Resource Q and S resin (Pharmacia, Uppsala, Sweden)

Generation of Transducible Tat Fusion Proteins

To produce genetic in-frame Tat fusion proteins, we constructed a bacterial expression vector, pTAT–HA, that contains an N-terminal 6-histidine leader followed by the 11-amino acid Tat protein transduction domain5 flanked by glycine residues for free bond rotation of the domain, a hemagglutinin (HA) tag, and a polylinker (Fig. 1A). As examples, pTat–HA vectors expressing full-length wild-type and mutant cDNAs from the p16INK4a tumor suppressor protein (Tat–p16, 20 kDa), human papilloma virus E7

Urea Denaturation of Tat Fusion Proteins

The exact mechanism of transduction across bilipid membranes is currently unknown; however, an analysis of Tat–p27Kip1 protein revealed that urea-denatured proteins elicit biological phenotypes more efficiently than soluble, correctly folded protein.11 We hypothesized that, because of reduced structural constraints, higher energy ΔG), denatured proteins may transduce more efficiently into cells than lower energy, correctly folded proteins. Once inside the cell, transduced denatured proteins

Solubilization of Tat Fusion Proteins into an Aqueous Buffer

To use the Tat fusion proteins in an aqueous environment, such as tissue culture media, rapid removal of the 8 M urea is required. This also achieves the goal of obtaining high energetic (ΔG), denatured Tat fusion proteins. Several choices for this procedure are described below. In our experience, the use of Mono Q or S ion-exchange chromatography yields superior results than rapid dialysis or utilization of a desalting column. However, we have generated transducible Tat fusion proteins by both

Transduction of Fluorescein-Labeled Tat Fusion Proteins into Cells

Tat fusion proteins can be labeled with fluorescein isothiocynate (FITC), which conjugates to lysine residues. Tat fusion protein (20–50 μg) is placed in a 300–μl reaction mix as per the manufacturer instructions for 2 hr at room temperature. The nonconjugated FITC is then removed from the FITC-labeled Tat protein by either gel-filtration chromatography or use of a PD-10 desalting column. We routinely use a PD-10 column equilibrated in PBS. Check the column fractions by Bio-Rad protein analysis

Biological Activities of Transduced Tat Fusion Proteins

Previously, p16INK4a and E1A proteins have been shown to bind Cdk6 and the retinoblastoma protein (pRb), respectively.17 Therefore, to analyze for in vivo biochemical function of transduced proteins, p16(-/-) Jurkat T cells were treated with 100 or 200 nM (final concentration) Tat–p16 protein during concomitant [35S]methionine labeling of cellular proteins. Cellular lysates were prepared and Tat fusion proteins were immunoprecipitated with anti-p16

Discussion

We have utilized the procedure described here to generate and transduce more than 50 urea-denatured proteins into ∼100% of all cells assayed thus far, including primary cells from blood, osteoclasts, bone marrow stem cells, and peripheral blood lymphocytes.11, 12, 13 The use of a genetic in-frame fusion combined with denaturation of the proteins achieves several goals, including isolation of the bulk of recombinant proteins that are usually present in inclusion bodies, increased efficiency of

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