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      Membrane penetrating peptides greatly enhance baculovirus transduction efficiency into mammalian cells

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          Research highlights

          ► Ligation of CTP with GP64 enhances baculovirus transduction into mammalian cells. ► Fusion of PTD with VP39 enhances baculovirus transduction into mammalian cells. ► CTP and PTD-carrying viruses improve the transduction of co-transduced baculoviruses. ► Virus entry and gene expression can be separate events in different cell types.

          Abstract

          The baculovirus group of insect viruses is widely used for foreign gene introduction into mammalian cells for gene expression and protein production; however, the efficiency of baculovirus entry into mammalian cells is in general still low. In this study, two recombinant baculoviruses were engineered and their ability to improve viral entry was examined: (1) cytoplasmic transduction peptide (CTP) was fused with baculovirus envelope protein, GP64, to produce a cytoplasmic membrane penetrating baculovirus (vE-CTP); and (2) the protein transduction domain (PTD) of HIV TAT protein was fused with the baculovirus capsid protein VP39 to form a nuclear membrane penetrating baculovirus (vE-PTD). Transduction experiments showed that both viruses had better transduction efficiency than vE, a control virus that only expresses EGFP in mammalian cells. Interestingly, vE-CTP and vE-PTD were also able to improve the transduction efficiency of a co-transduced baculovirus, resulting in higher levels of gene expression. Our results have described new routes to further enhance the development of baculovirus as a tool for gene delivery into mammalian cells.

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          Most cited references25

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          A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.

          Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
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            Baculovirus as versatile vectors for protein expression in insect and mammalian cells

            Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
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              Cell-penetrating peptides: mechanism and kinetics of cargo delivery.

              Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The only common feature of these peptides appears to be that they are amphipathic and net positively charged. The mechanism of cell translocation is not known but it is apparently receptor and energy independent although, in certain cases, translocation can be partially mediated by endocytosis. Cargoes that are successfully internalized by CPPs range from small molecules to proteins and supramolecular particles. Most CPPs are inert or have very limited side effects. Their penetration into cells is rapid and initially first-order, with half-times from 5 to 20 min. The size of smaller cargoes does not affect the rate of internalization, but with larger cargoes, the rate is substantially decreased. CPPs are novel vehicles for the translocation of cargo into cells, whose properties make them potential drug delivery agents, of interest for future use.
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                Author and article information

                Contributors
                Journal
                Biochem Biophys Res Commun
                Biochem. Biophys. Res. Commun
                Biochemical and Biophysical Research Communications
                Elsevier
                0006-291X
                1090-2104
                8 January 2011
                11 February 2011
                8 January 2011
                : 405
                : 2
                : 297-302
                Affiliations
                [a ]Institute of Biotechnology, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan, ROC
                [b ]Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC
                Author notes
                [* ]Corresponding authors: Fax: +886 2 27826085. mbycchao@ 123456imb.sinica.edu.tw liucat_2@ 123456yahoo.com
                Article
                S0006-291X(11)00051-9
                10.1016/j.bbrc.2011.01.032
                7092845
                21219863
                59614d3a-b7e7-45dc-84a5-4bd3e8fd5069
                Copyright © 2011 Elsevier Inc. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 18 December 2010
                Categories
                Article

                Biochemistry
                baculovirus,transduction,cytoplasmic transduction peptide,protein transduction domain,mammalian cells

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