Production of Cell Lines Secreting TAT Fusion Proteins
Center for Anatomy and Functional Morphology and Department of Pathology, Mount Sinai School of Medicine, New York, New York (TB); Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York, New York (ESG); and Brookdale Department of Molecular Cell and Developmental Biology, Mount Sinai School of Medicine, New York, New York (SCH)
Correspondence to: Tibor Barka, MD, Center for Anatomy and Functional Morphology, Box 1007, Mount Sinai School of Medicine, New York, NY 10029. E-mail: Tibor.Barka{at}mssm.edu
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Summary |
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Key Words: TAT fusion proteins green fluorescent protein red fluorescent protein mammalian expression vector
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Introduction |
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Studies of the mechanism of transduction of proteins led to the identification of protein transduction domains (PTDs). The most widely studied of these sequences, in addition to TAT, are the Drosophila antennapedia peptide (Derossi et al. 1994) and the Herpes simplex virus VP22 protein (Elliott and O'Hare 1997
). However, it is now recognized that other proteins and synthetic peptides have similar translocating properties (Derossi et al. 1998
; Lindgren et al. 2000
; Schwartz and Zhang 2000
; Ford et al. 2001
; Ho et al. 2001
; Bogoyevitch et al. 2002
; Lindsay 2002
; Mai et al. 2002
; Park et al. 2002
; Futaki et al. 2003
), some of which (e.g., the 9-mer of D-arginine) (Wender et al. 2000
) or polylysine (Mai et al. 2002
), are more effective in cellular uptake than Tat4957.
The technology of generating TAT fusion proteins (BeckerHapak et al. 2001) requires the synthesis of the fusion protein in which the TAT transduction domain (amino acids 4757 of HIV Tat, termed TAT) is linked to the molecule of interest, using a bacterial expression vector. In general, the TAT peptide is also linked to a tag to facilitate its subsequent purification. The purified recombinant fusion protein can be added directly to mammalian cells in culture or injected in vivo into an animal (Schwarze et al. 1999
). We have shown that retrograde ductal injection of a fusion protein (TATß-galactosidase) into rat salivary glands provides targeted delivery and that epithelial and mesenchymal cells of developing mouse submandibular glands in organ culture can be transduced by the same fusion protein (Barka et al. 2000
). The technique outlined is generally successful but laborious. Furthermore, if a sustained effect of the TAT fusion protein is required, repeated exposure in vitro or administration in vivo is necessary.
We conceived an alternative technology that offers certain advantages in applying transduction techniques mediated by TAT and other PTDs. To this end, we constructed mammalian expression vectors expressing and secreting TAT fusion proteins, transfected cultured cells with such vectors, and established stable transformed cell lines. The TAT fusion protein is secreted by such cells into the culture medium, which can be added directly to other cells in culture. Cells can also be co-cultured with secreting transformed cells, thus exposing them continuously to the TAT fusion protein. If required, the TAT fusion protein can also be purified from the spent culture medium by conventional techniques. Furthermore, we foresee that such transformed cells could be a sustained source of PTD fusion peptides and other macromolecules in vivo. To demonstrate the feasibility of this approach, we describe, as a prototype, the construction of a mammalian expression vector designed for stable expression and secretion of TATgreen fluorescent protein (TATEGFP) in mammalian hosts and the use of this vector in transduction experiments.
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Materials and Methods |
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Transfection
We transfected CHO-K1 cells using FuGENE 6 Transfection Reagent (Roche Applied Science; Indianapolis, IN) in serum-containing medium according to the manufacturer's instructions. The transfection efficiencies with FuGENE Reagent:DNA ratios (µl and µg, respectively) of 3:1 and 6:1 were similar and were about twice as great as that obtained with a ratio of 3:2. We carried out transfections with three plasmids: pEGFPN1, pCMVDsRed-Express (BD Biosciences Clontech; Palo Alto, CA), and pTATGFP. We selected stable transformed cell lines, designated as GFPN, CHORED and CHOGFP, respectively, using 1 mg/ml Geneticin (GFPN and CHORED) or 400 µg/ml of Zeocin (CHOGFP) starting 48 hr after transfection. Zeocin and Geneticin sensitivities of the parent CHO-K1 cells were determined in preliminary experiments. The transformed cells were maintained in the same concentrations of the antibiotics.
Tissue Cultures and Cell Lines
CHO-K1 Chinese hamster ovary cells (ATCC, #CCL-61) were grown in F12-K medium (American Type Culture Collection; Manassas, VA) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 µg/ml streptomycin. CHOGFP cells were grown in the same medium supplemented with 400 µg/ml of Zeocin. In the case of CHORED cells the medium was supplemented with 1 mg/ml of Geneticin. CHOGFP and CHORED cells were cloned using cloning cylinders or by limiting dilutions. Clones were selected on the basis of expression of the fluorescent protein. For serum-free culture, CD CHO A Medium (GIBCO Invitrogen; Grand Island, NY), supplemented with 4 mM L-glutamine and antibiotics, was used. This medium is designed for the growth of anchorage-dependent CHO-K1 cells.
Viability Assay
We assayed the viabilty of CHGGFPPOS cells using the LIVE/DEAD Viability/Cytotoxicity Assay Kit (L3224) (Molecular Probes; Eugene, OR) following the protocol of the manufacturer. The two-color fluorescence assay is based on the enzymatic conversion by ubiquitous esterases of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, and the permeability of dead but not living cells by ethidium bromide. We grew the cells on coverslips and incubated with the reagent for 30 min at room temperature. According to our preliminary experiments, for this cell line the optimal concentrations of ethidium bromide and calcein AM are both 2 µM. The cells were killed by 30-min treatment with 70% ethanol. We viewed the preparations with a Zeiss Axioskop microscope and photographed them using the same exposure time for all preparations.
Exposure of Cultured Cells to Spent Media of TATGFP-secreting Cells
CHOGFPClone 6 cells were cultured in 20 ml F12-K medium supplemented with 10% FBS, penicillin/streptomycin, and 400 µg/ml Zeocin in 10-cm petri dishes. When the cultures were near confluence, the medium was replaced with 24 ml fresh medium without Zeocin. The medium was collected after 3 hr of incubation at 37C and centrifuged at 3000 rpm for 5 min. The supernatant was used immediately or kept at -20C.
To test the transduction of TATGFP fusion protein, we plated CHOREDClone 15 cells onto coverglasses in 35-mm dishes, 22.5 x 105 cells per dish in 2 ml medium. Next day, we replaced the medium with 2 ml spent medium prepared as described above. In general, we have examined the cells under a fluorescence or confocal microscope without fixation. However, we also fixed some cultures after 30 min, 1, 2, 4, or 24 hr of exposure and examined them under a fluorescence microscope.
Western Blots
For Western blots, we cultured CHOGFPC16 or CHOGFPPOS cells in serum-free medium. We harvested the medium from cultures at near confluence, concentrated it by Amicon Ultra Filter Devices (30,000 MWCO), separated the proteins by SDS-PAGE (10%), and electroblotted them onto PVDF membranes. The Western blots also included recombinant EGFP (BD Biosciences Clontech). For immunostaining, we used a monoclonal antibody to EGFP [BD Living Colors A.v. Monoclonal Antibody (JL-8); BD Biosciences Clontech] at a dilution of 1:2500 and a peroxidase chemiluminescent method (ECL Advance Western Blotting Detection Kit; Amersham Biosciences, Piscataway, NJ) with the secondary antibody, peroxidase-labeled anti-mouse IgG, at a dilution of 1:10,000. For blocking and for the dilutions of the antibodies we used 1 x TBS/Casein Blocker (Bio-Rad; Hercules, CA).
Fluorescent Microscopy
We examined most cultures without fixation with a confocal microscope [Leica TCS-SP (UV)] or a Zeiss Axiphot fluorescence microscope. For observations of living cells, they were grown in Glass Bottom Microwell Dishes (MatTek; Ashland, MA). However, some cultures were observed after fixation. To this end, cultured cells were rinsed twice with PBS, fixed in 2% formaldehyde [formaldehyde, (methanol free) 10% ultrapure EM grade; Polysciences, Warrington, PA, diluted with PBS)] for 30 min, and washed in PBS twice for 5 min. The cultures were mounted in Vectashield Hard Set Mounting Medium (Vector Laboratories; Burlingame, CA). The microscopic images of living and fixed cells were comparable.
Flow Cytometry
For sorting of cells expressing GFP or DsRed we used a FACSVantage SE high-speed cell sorter (Becton Dickinson; Mountain View, CA) (Flow Cytometry Shared Research Facility, Mount Sinai School of Medicine). Cells positive for GFP or DsRed were then cultured in F12-K1 medium supplemented with 10% FSB and antibiotics.
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Results |
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CHO-K1 cells were transfected with pEGFP-N1 or pCMV-DsRed-Express at high efficiency (Figures 1A and 1D). The transfection efficiency with pTATGFP was less. We established stable transformed cell lines expressing EGFP, DsRed, or TATEGFP, respectively. In the case of cells transfected with pEGFP-N1 or pCMV-DsRed-Express, the selective medium included 1 mg/ml of Geneticin. In the case of cells transfected with pTATTAG, the selection was based on resistence to 400 µg/ml of Zeocin. However, in further experiments we used only two cell lines expressing TATEGFP, designated CHOGFP, or DsRed, designated CHO-RED, respectively. We cloned both CHOGFP and CHORED cells and selected clones on the basis of the percentage of cells expressing the fluorescent protein and the level of expression. However, we applied these criteria without accurate quantitation. In cultures of the two clones selected, CHOGFPC16 and CHOREDCl15, not all cells express the corresponding fluorescent protein and the level of expression varies greatly among the cells (Figures 1G and 1I). The reason for this variation is not known. These clones were maintained in the selective medium for several months without apparent change in the expression of EGFP or DsRed.
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Viability Assays of CHOGFPPOS Cells
By using a two-color fluorescence assay, we estimated the viabilty of CHOGFPPOS cells used to collect spent media. In sparse cultures and in cultures near confluence, >99% of the cells were viable. When cells killed by fixation with 70% ethanol were stained, all cells showed intense red fluorescence in the nuclei and only faint fluorescence in the cytoplasm, an indication of the proper concentration (2 µM) of ethidium bromide in the assay reagent (Figure 4A). In such preparations, no green fluorescence was detected, again indicating the appropriateness of the reagent's calcein AC concentration, 2 µM (Figure 4B). Cultures of CHOGFPPOS cells showed intense green fluorescence of living cells (Figure 4C), and only occasional dead cells in the red channel (Figure 4D). In overgrown cultures, the proportion of dead cells increased but was still less than 5% (data not shown).
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Western blots of concentrated serum-free spent media of CHOGFP cells, prepared by using a monoclonal antibody to EGFP, revealed a single band of protein with the same migration in SDS-PAGE as rEGFP (Figure 5). The apparent molecular weights of rEGFP and the reacting protein of the concentrate were approximately 60 kD, suggesting that they may represent dimers of EGFP. However, the nature of these bands was not investigated.
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Discussion |
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The technique of producing TAT fusion proteins, and PTD fusion proteins in general, requires the synthesis and purification of such proteins using bacterial expression systems (VoceroAkbani et al. 2000; BeckerHapak et al. 2001
). As an alternative, we have developed a methodology based on the use of a mammalian expression vector, pSecTag2. This vector is designed for high-level expression and secretion of proteins by transient or stable integrants. As a model, we developed stable transformed cell lines, derived from CHO cells, expressing and secreting TATEGFP. Although we have applied the same technology to generate cell lines that express and secrete TATß-galactosidase, GFP offers high sensitivity and ease of detection in fixed and living cells to illustrate the advantages of this technique.
CHO-K1 cells transfected with pEGFP-N1 or pTATGFP express EGFP. Similarly, cells transfected with pCMVDsRedExpress express the red fluorescent protein DsRed. We have obtained cell lines of stable integrants by exposing the transfected cells to Zeocin (CHOGFP) or Geneticin (CHORED). Cloning of Zeocin-resistant CHOGFP cells resulted in clones in which only a fraction of the cells expressed EGFP. Of 10 clones examined, clone 6 showed the highest percentage of cells, approximately 20%, expressing GFP. The level of expression varied from cell to cell. The reason for this variation in expression of the stable transgene is not known but is probably, at least in part, caused by the inherently stochastic nature of gene expression. Stochastic mechanisms in gene expression operate in both prokaryotes and eukaryotes and may explain the phenotypic variations in isogenic populations of cells (McAdams and Arkin 1997; Elowitz et al. 2002
; Ozbudak et al. 2002
; Blake et al. 2003
). Other mechanisms, such as the conversion of GFP to the fluorescent form (Heim et al. 1994
) and the equilibrium between synthesis of the protein and its secretion, are probably also operational. Non-uniform production of GFP among HEK293 cells transfected with a vector coding for a mutant of GFP has been described (Malek and Khaledi 1999
).
Analysis by flow cytometry confirmed that only approximately 20% of CHO-GFP-Cl6 cells expressed GFP. Sorting of GFP+ cells and their expansion led to a cell line in which approximately 98% of the cells expressed EGFP. Whether the strategy of first cloning stable transformed cells by conventional cloning technique and subsequent "cloning" by sorting of cells expressing the transgene is more efficient than cloning by flow cytometry alone remains to be investigated.
A similar cell-to-cell variation was evident in CHORED cells. This conspicuous cell variation in the level of DsRed fluorescence existed even in the CHOREDPOS cell line that was generated by sorting DsRed-expressing cells.
CHOGFP cells not only express but also secrete GFP. This was established by Western analysis of media in which the cells were cultured and by exposing cells to spent media. For the latter experiment, we used a cell line (CHORED) expressing Ds-Red fluorescent protein. This strategy provided unequivocal proof of secretion and transduction of TATEGFP by excluding the presence of autofluorescent material in the recipient cells. Viability studies indicated that approximately 99% of CHOGFPPOS cells that provided the spent media were viable, obviating the possibility that TATGFP was released from dead cells and not secreted by living cells.
In summary, the use of a mammalian secretory system to generate PTD fusion proteins offers certain advantages: relative ease of preparing the PTD fusion proteins in soluble form, which can be added directly to cultured cells, and potential post-translational modifications, such as glycosylation and formation of disulfide bonds. However, such proteins can also be purified by conventional techniques for in vitro or in vivo applications, providing a stable source of such proteins in vitro and possibly in vivo. We foresee therapeutic applications of this technique in which the parent cells transfected with constructs coding for the PTD fusion proteins could conceivably be the patient's own cells. This system is also adaptable to the study of autocrine regulatory mechanisms by transfecting cells with constructs coding for PTD-fused putative autocrine peptides.
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Acknowledgments |
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We are grateful to Ms H. van der Noen for excellent assistance, Dr I. Karpichev for advice in cloning, Dr C. Iomini for stimulating discussions, and Mr P.T. Carman for assistance with microscopy.
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Footnotes |
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Literature Cited |
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