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Protein Science (2001), 10:2138-2139.
Copyright © 2001 The Protein Society

FOR THE RECORD

Heparin binding by the HIV-1 tat protein transduction domain

Susanna Hakansson, Amy Jacobs and Michael Caffrey

Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60612, USA

Reprint requests to: Michael Caffrey, Department of Biochemistry, University of Illinois at Chicago, 1819 W. Polk St., Chicago, IL 60612; email: caffrey{at}uic.edu; fax: (312) 413-0364.

(RECEIVED June 12, 2001; FINAL REVISION June 15, 2001; ACCEPTED June 15, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.23401.


   Abstract
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 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
The protein transduction domain from the HIV-1 tat protein (termed PTD-tat) has been fused to the C-terminus of a model cargo protein, the IgG binding domain of streptococcal protein G. We demonstrate that PG-Ctat (PTD-tat fused to the C-terminus of protein G) binds to a heparin affinity column. PG-Ctat binds with relatively high affinity, as shown by its elution at 1.6 M NaCl. The heparin binding properties of PTD-tat are consistent with the idea that heparan sulfate, an analog of heparin found at the cell surface, plays a role in the translocation of PTD-tat fusions. We suggest that the heparin-binding properties of PTD-tat can be exploited for purification of PTD-tat fusions in the absence of affinity tags.

Keywords: Heparan sulfate; heparin; HIV; protein transduction; tat

Abbreviations: IPTG, isopropyl-b-D-thiogalactopyranoside • PG, IgG binding domain of streptococcal protein G • PG-Ctat, HIV-1 tat PTD domain fused to C-terminus of PG • PTD, protein transduction domain • PTD-tat, HIV-1 tat PTD domain


   Introduction
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 Abstract
 Introduction
Results and Discussion
 Materials and methods
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A small region of HIV-1 tat, termed protein transduction domain or PTD-tat, has been shown to mediate transfer of proteins across biological membranes in cell culture and in living animals (Nagahara et al. 1998; Schwarze et al. 1999). Moreover, it has been established that PTD-tat can be used for intracellular delivery of DNA and drug compounds in addition to proteins (for review, see Schwarze and Dowdy 2000). Consequently, PTD-tat can be envisioned as a delivery tool for a wide variety of therapeutic agents to diverse cell types. The mechanism of PTD-tat-mediated translocation is unknown, but is thought to not involve endocytosis (Schwarze and Dowdy 2000). It has been shown that the HIV-1 tat protein, which contains PTD-tat, binds to heparin (Rusnati et al. 1997) and that internalization of the tat protein requires the presence of cell-surface heparan sulfate proteoglycans (Tyagi et al., 2001). The present findings demonstrate that the PTD-tat sequence binds to immobilized heparin, a close structural analog of heparan sulfate (Yanagishita and Hascall 1992) that may mediate translocation in vivo when fused to a model cargo protein. We suggest that the heparin-binding properties of PTD-tat can be exploited as a general scheme for the purification of PTD fusions.


   Results and Discussion
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 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
For the study of PTD-tat, we chose to fuse it to the C-terminus of the IgG-binding domain of streptococcal protein G (PG), which is well characterized (Gronenborn et al. 1991; Barchi et al. 1994; Huth et al. 1997). The ability of the fusion protein, PG-Ctat, to bind to heparin was assayed by heparin affinity chromatography. Heparin affinity chromatography was performed using a HiPrep 16/10 Heparin FF column (16 mm X 100 mm, Amersham Pharmacia Biotech, Piscataway, NJ) in conjunction with a BioCad Sprint chromatography system (PE Biosystems, Foster City, CA) at room temperature. The chromatograms of purified PG and PG-Ctat are shown in Figure 1Go. In the absence of PTD-tat, PG does not bind to the heparin affinity column under loading buffer conditions, as shown by its elution before the salt gradient. In contrast, PG-Ctat clearly binds to the heparin affinity column and elutes at ~1.6 M NaCl, suggesting that it binds rather strongly to heparin. Since the PG moiety does not bind, the PTD-tat sequence can be inferred to bind to heparin with high affinity. Previous studies of the HIV-1 tat protein (residues 1–86) fused to the C-terminus of glutathione S-transferase have shown that it binds to heparin affinity columns and that it elutes at ~1.5 M NaCl (Rusnati et al. 1997). The present work is the first demonstration that the 11 residues of the PTD-tat sequence, within the context of a cargo protein, bind to a heparin affinity column with approximately the same affinity as the tat protein. PTD-tat possesses eight basic residues, which suggests that its interaction with heparin is at least partially mediated by electrostatic interactions between the basic amino acid sidechains of PTD-tat and the sulfate moieties of heparin. As noted by Tyagi et al. (2001), heparin-like molecules are widely distributed on the surface of cells in the form of heparan sulfate proteoglycans. Thus, the interaction between PTD-tat and the immobilized heparin may indeed mimic the physiological interaction. Finally, we note that the heparin-binding properties can be exploited as a general purification scheme for proteins containing PTD-tat, which are exciting new tools for biology and medicine. Importantly, the use of heparin affinity chromatography obviates the need for other affinity labels such as histidine or glutathione S-transferase tags, which have previously been used (c.f. Rusnati et al. 1997; Schwarze et al. 1999).



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  		Fig. 1. Chromatograms of PG (dashed line) and PG-Ctat (solid line) binding to a heparin affinity column. The flow rate was set to 5 mL/min. The injection volume was 50 µL. The elution conditions were 8 min of 20 mM Tris-HCl/pH 8.0, 150 mM NaCl followed by 20 mM Tris-HCl/pH 8.0 and a 150 mM to 2000 mM NaCl gradient over 8 min. The percentage of elution buffer is shown as a dotted line.

 

   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
The PTD-tat sequence was attached to the C-terminus of a model protein, the IgG-binding domain of streptococcal protein G (Huth et al. 1997) by standard molecular biology techniques to form PG-Ctat. Overlapping oligonucleotides containing the PTD-tat sequence and overhanging bases corresponding to the NheI and XhoI sites were synthesized (IDT, Coralville, IA). The overlapping oligonucleotides were mixed, annealed, and ligated into the NheI/XhoI site of pGEV1, which is an expression vector for protein G and contains NheI and XhoI sites at the C-terminus (Huth et al. 1997). The resulting PG-Ctat construct contained 77 residues: the 56 residues of PG, a 6-residue linker region (PGGPAS), PTD-tat (YGRKKRRQRRR), and a 4-residue cloning aact at the C-terminus (GGGG). Protein expression of PG and PG-Ctat was achieved in E. coli strain BL21. For large-scale preparations, the appropriate strains were grown in 2L batches of LB media supplemented with 50 µg/mL ampicillin at 37°C. Protein expression was induced at the full exponential growth phase (OD600 ~0.4) by the addition of IPTG to a final concentration of 0.8 mM; cell growth was continued for 4 h and the cells were harvested by centrifugation. The recombinant proteins were found primarily in the inclusion bodies. The general purification scheme proceeded by first resuspending the cells in 100 mM Tris-HCl/pH 7.3, 10 mM EDTA and then disrupting cellular membranes by sonication (5 x 1 min). Inclusion bodies were isolated by centrifugation, and the recombinant proteins were solubilized in 8M Gdn-HCl, 100 mM Tris/pH 7.3. The recombinant protein was then purified by size exclusion chromatography (Sephacryl-200 column, Amersham Pharmacia Biotech) in 4M Gdn-HCl, 100 mM Tris-HCl/pH 7.3 at 4°C. In the next step, the partially purified protein was loaded onto a reverse phase column (Poros R2, PE Biosystems) equilibrated in 0.1% TFA and eluted with a 0–70% acetonitrile gradient in 0.1% TFA at room temperature. The purified proteins were then diluted to ~0.1 mg/mL and refolded by dialysis against 100 mM Tris-HCl/pH 7.3, 150 mM NaCl at 4°C overnight with 3 buffer changes. The proteins were filtered, concentrated by ultrafiltration (YM3, Amicon), and stored at -70°C.


   Acknowledgments
 
This work was supported by startup funds from the University of Illinois at Chicago.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Barchi, J., Grasberger, B., Gronenborn, A., and Clore, G. 1994. Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy. Protein Sci. 3: 15–21.[Abstract]

Gronenborn, A., Filpula, D., Essig, N., Achari, A., Whitlow, M., Wingfield, P., and Clore, G. 1991. A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253: 657–661.[Abstract/Free Full Text]

Huth, J., Bewley, C., Jackson, B., Hinnebusch, A., Clore, G., and Gronenborn, A. 1997. Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR. Protein Sci. 6:2359–2364.[Abstract]

Nagahara, H., Vocero-Akbani, A., Snyder, E., Ho, A., Lathham, D., Lissy, N., Becker-Hapak, M., Ezhevsky, S., and Dowdy, S. 1998. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27(Kip1) induces cell migration. Nat. Med. 4: 1449–1452.[CrossRef][Medline]

Rusnati, M., Doltrini, D., Oreste, P., Zoppetti, G., Albini, A., Noonan, D., Fagagna, F., Giacca, M., and Presta, M. 1997. Interaction of HIV-1 tat protein with heparin. J. Biol. Chem. 272: 11313–11320.[Abstract/Free Full Text]

Schwarze, S. and Dowdy, S. 2000. In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21: 45–48.[CrossRef][Medline]

Schwarze, S., Ho, A., Vocero-Akbani, A., and Dowdy, S. 1999. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science 285: 1569–1572.[Abstract/Free Full Text]

Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. 2001. Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J. Biol. Chem. 276:3254–3261.[Abstract/Free Full Text]

Yanagishita, M. and Hascall, V. 1992. Cell surface heparan sulfate proteoglycans. J. Biol. Chem. 267:9451–9454.[Free Full Text]


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