Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein.
ABSTRACT Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.
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ABSTRACT: Secreted and intracellular proteins including antibodies, cytokines, major histocompatibility complex molecules, antigens, and enzymes can be redirected to and anchored on the surface of mammalian cells to reveal novel functions and properties such as reducing systemic toxicity, altering the in vivo distribution of drugs and extending the range of useful drugs, creating novel, specific signaling receptors and reshaping protein immunogenicity. The present review highlights progress in designing vectors to target and retain chimeric proteins on the surface of mammalian cells. Comparison of chimeric proteins indicates that selection of the proper cytoplasmic domain and introduction of oligiosaccharides near the cell surface can dramatically enhance surface expression, especially for single-chain antibodies. We also describe progress and limitations of employing surface-tethered proteins for preferential activation of prodrugs at cancer cells, imaging gene expression in living animals, performing high-throughput screening, selectively activating immune cells in tumors, producing new adhesion molecules, creating local immune privileged sites, limiting the distribution of soluble factors such as cytokines, and enhancing polypeptide immunogenicity. Surface-anchored chimeric proteins represent a rich source for developing new techniques and creating novel therapeutics.Medicinal Research Reviews 06/2008; 28(6):885-928. · 8.13 Impact Factor
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ABSTRACT: The use of T cell receptors (TCRs) as potential therapeutic agents provides an opportunity to target a greatly expanded array of antigens, compared to those now targeted with monoclonal antibodies. With the advent of new display technologies and TCR formats for in vitro engineering, it should be possible to generate high-affinity TCRs against virtually any peptide antigen that is shown to bind to a major histocompatibility complex (MHC) molecule (e.g. peptides derived from viral antigens or from self proteins that are associated with the transformed phenotype). What remains, however, are challenges associated with effective targeting of very low numbers of cell surface antigens (pepMHC), fewer than the case for conventional monoclonal antibody-based therapies. This hurdle might be overcome with the attachment of more effective payloads for soluble TCR approaches, or by using TCR gene transfer into T cells that can then be adoptively transferred into patients. There is considerable work to be done on the physiological aspects of either approach, including pharmacokinetic studies in the case of soluble TCRs, and T cell trafficking, persistence, and autoreactivity studies in the case of adoptively transferred T cells. As with the field of monoclonal antibodies, it will take time to explore these issues, but the potential benefits of TCR-based therapies make these challenges worth the effort.Biomolecular Engineering 11/2007; 24(4):361-73. · 3.17 Impact Factor
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ABSTRACT: Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.Advanced Drug Delivery Reviews 01/2007; 58(15):1622-54. · 12.71 Impact Factor