Negatively charged liposome as a potent inhibitor of post-translation during in vitro synthesis of green fluorescent protein

Bui, Huong Thi; Umakoshi, Hiroshi; Suga, Kazuyoshi; Nishida, Masaru; Shimanouchi, Toshinori; Kuboi, Ryoichi

doi:10.1016/j.bej.2009.05.002

Cited by 12 publications

(6 citation statements)

References 29 publications

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“…On the other hand, the POPC/DOTAP(70/30) liposome drastically inhibited it to 37%. Our previous studies investigated the effect of liposomes on in vitro GFP expression using a cell-free system ( 9–11 , 13 ), where the POPC/Ch(70/30) liposomes could recognize tRNA or mRNA, then induce their conformational change (destabilization of RNAs towards heat stress). Since the folding of mRNA affected its translation ( 15 ), a suitable destabilization of mRNA was found to enhance mRNA translational activity.…”

Section: Resultsmentioning

confidence: 99%

“…Details of the mechanisms were investigated, focusing on each elemental step (transcription, translation and folding), with the finding that the liposomes significantly affected the translation step; the POPC/Ch(70/30) liposome enhanced the polypeptide synthesis, while the cationic liposomes inhibited it ( 11 ). The anionic liposomes did not affect the translation; they rather inhibited the folding of the GFP peptide ( 9 , 13 ). Therefore, the heterogeneous liposomes or the cationic liposomes would have significant effects on the translation step.…”

Section: Introductionmentioning

confidence: 96%

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Conformational change of single-stranded RNAs induced by liposome binding

Suga

Tanabe

Tomita

et al. 2011

Nucleic Acids Research

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The interaction between single-stranded RNAs and liposomes was studied using UV, Fourier Transform Infrared spectroscopy (FTIR) and Circular Dichroism spectroscopy (CD). The effect of the surface characteristics of liposomes, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modified with cholesterol (Ch) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), on the liposome–RNA interaction was investigated. The fluorescence of 6-(p-toluidino)naphthalene-2-sulfonate (TNS) embedded in the liposome surface (ε = 30–40) was decreased in the presence of tRNA, suggesting that single-stranded tRNA could bind onto the liposome. The dehydration of –PO2− –, guanine (G) and cytosine (C) of tRNA molecules in the presence of liposomes suggested both an electrostatic interaction (phosphate backbone of tRNA and trimethylammonium group of POPC, DOTAP) and a hydrophobic interaction (guanine or cytosine of tRNA and aliphatic tail of lipid). The tRNA conformation on the liposome was determined by CD spectroscopy. POPC/Ch (70/30) maintained tRNA conformation without any denaturation, while POPC/DOTAP(70/30) drastically denatured it. The mRNA translation was evaluated in an Escherichia coli cell-free translation system. POPC/Ch(70/30) enhanced expression of green fluorescent protein (GFP) (116%) while POPC/DOTAP(70/30) inhibited (37%), suggesting that the conformation of RNAs was closely related to the translation efficiency. Therefore, single-stranded RNAs could bind to liposomal membranes through electrostatic and hydrophobic attraction, after which conformational changes were induced depending on the liposome characteristics.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

Conformational change of single-stranded RNAs induced by liposome binding

Suga

Tanabe

Tomita

et al. 2011

Nucleic Acids Research

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“…35 The amount of GFP evaluated by western blotting reflects the net expression of synthesized protein (that is, steps (i) and (ii)). To investigate the effect of CHP nanogels on the downstream processes (that is, step (iii)), the formation of correctly folded, mature GFP was analyzed by fluorescence spectroscopy, as shown in Figure 2.…”

Section: Resultsmentioning

confidence: 99%

Polysaccharide nanogel–cyclodextrin system as an artificial chaperone for in vitro protein synthesis of green fluorescent protein

Sasaki

Nomura

Sawada

et al. 2010

Polym J

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Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/ translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-b-cyclodextrin (M-b-CD). The CHP nanogels dissociate on complexation with M-b-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel-protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-b-CD was comparable to that of spontaneous folding in the absence of CHP/M-b-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins.

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“…A model biomembrane, liposome, has previously been reported to induce a variety of potential functions under variable environmental conditions, including a molecular chaperone-like function, 7 the translocation of proteins across membranes, 8 membrane fusion, 9 and LIPOzyme functions 10 - 14 under appropriate stress conditions. In our series of works, it has been previously reported that liposomes can affect the in vitro gene expression system 15 - 20 , 31 including a series of steps (i.e. (i) transcription (DNA to RNA), (ii) translation (mRNA to polypeptide), and (iii) folding (conformational change of polypeptide), herewith defined as “Biomembrane Interference.” Especially in the case of oxidative stress, it has also been reported that variations in the characteristics of the liposome membrane can occur through the oxidation of the unsaturated part inside a phospholipid bilayer by a reactive oxygen species.…”

Section: Introductionmentioning

confidence: 99%

Oxidative Stress Can Affect the Gene Silencing Effect of DOTAP Liposome in an In Vitro Translation System

Umakoshi¹,

Tanabe

Suga

et al. 2011

Int. J. Biol. Sci.

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Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.

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Negatively charged liposome as a potent inhibitor of post-translation during in vitro synthesis of green fluorescent protein

Cited by 12 publications

References 29 publications

Conformational change of single-stranded RNAs induced by liposome binding

Conformational change of single-stranded RNAs induced by liposome binding

Polysaccharide nanogel–cyclodextrin system as an artificial chaperone for in vitro protein synthesis of green fluorescent protein

Oxidative Stress Can Affect the Gene Silencing Effect of DOTAP Liposome in an In Vitro Translation System

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