Production of Cell Lines Secreting TAT Fusion Proteins

Barka, Tibor; Gresik, Edward S.; Henderson, Scott C.

doi:10.1177/002215540405200405

Cited by 18 publications

(19 citation statements)

References 29 publications

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“…Antibodies usually need to be secreted by the producing organism. Although a stable CHO-K1 cell line that secretes TAT-EGFP has been reported, 157 there are also hints at problems with secretion of TAT-fusion proteins. In a study that compared different TAT-fusion proteins (TAT-EGFP, TATsrIκBα, TAT-RBD), the major fraction of fusion proteins was found in the cell lysate, and only a small amount of the fusion proteins was present in the supernatant.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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“…LIFRα-CT3, as reported in our previous research, was observed to have the capacity to activate signal transducer and activator of transcription3 (STAT3) in HL-60 cells by transfection, to initiate LIF-related intracellular signaling, and to facilitate both increasing differentiation and decreasing proliferation (5,26-28); however, transfection or virus-mediated gene delivery may cause an irreversible genetic modification, which makes these deliveries significantly unacceptable for possible clinical practice (29,30). Peptide-based cell delivery systems are greatly expanded by the recognition of PTDs and synthetic peptides with translocation properties (14,31,32). In the present study, we have shown that the TAT-CT3-cMyc fusion protein can be expressed in eukaryotic system CHO cells, secreted into the medium, and efficiently purified.…”

Section: Discussionmentioning

confidence: 99%

“…In general, the TAT fusion protein is also linked to some sort of tag so as to facilitate its subsequent purification. The purified recombinant fusion protein could be directly added to mammalian cells in culture or injected in vivo into an animal (14). The above technique is generally highly applicable but laborious; in addition, a protein that is derived from a prokaryotic expression system is potentially more limited by its lack of splicing and the associated post-transcription processing systems or post-translation modifying systems in comparison to eukaryotic expression systems.…”

Section: Introductionmentioning

confidence: 99%

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

Sun

Xiong

Lü

et al. 2012

Braz J Med Biol Res

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The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

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“…It has previously been shown that secreted TAT fusion proteins are able to transduce target cells but with very low efficiency. 16 We speculated that this maybe due to the presence of two furin cleavage sites within TAT-PTD which could result in the removal of the essential sequences from TAT-PTD by endogenous furin. Endogenous furin levels can vary between cell lines resulting in differences of furin-mediated processing.…”

Section: Discussionmentioning

confidence: 99%

“…17 Depending on the cell type used for secretion, small amounts of the fusion proteins may contain intact PTD and hence result in the inefficient transduction of target cells observed in the previous study. 16 However, the majority of the secreted fusion proteins are likely to lack TAT-PTD and therefore are unable to enter target cells. In this article we report novel modifications to the TAT peptide generating TATκ, which is resistant to furin cleavage and hence highly transducible.…”

Section: Discussionmentioning

confidence: 99%

Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

et al. 2009

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The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATkappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATkappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

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Production of Cell Lines Secreting TAT Fusion Proteins

Cited by 18 publications

References 29 publications

Targeting antibodies to the cytoplasm

Targeting antibodies to the cytoplasm

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

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