title: Effect of a Flexible Linker on Recombinant Expression of Cell-Penetrating Peptide Fusion Proteins and Their Translocation into Fungal Cells.
abstract: Cell-penetrating peptides (CPPs) are a class of small peptides that are able to cross cell membranes via direct translocation or endocytosis. They have been widely used to deliver tethered bioactive molecules to cells, but recombinantly producing CPPs as fusions to protein cargo leads to low yields. We used Escherichia coli cells to recombinantly produce genetic fusions of NPFSD (derived from a yeast endocytosis signal) and pVEC (derived from a murine vascular endothelium cadherin) to the N-terminus of green fluorescent protein (GFP) with and without a flexible glycine-serine linker between the CPP and GFP. The flexible linker improved the expression of the NPFSD construct and the pVEC construct, resulting in a 24.5 % improvement in yield for the NPFSD fusion and a 50.0 % improvement in yield for the pVEC fusion. The linker did not diminish the ability of the fusions to translocate into the fungal pathogen Candida albicans, and the translocation of the NPFSD constructs actually increased by 58 % at 10 min. Moreover, the toxicity of the fusions towards C. albicans was not affected by the incorporation of the linker. These results illustrate the utility of including a linker for CPP-cargo fusions and the potential of NPFSD and pVEC fusions for use in delivering protein cargo to C. albicans.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD, 20742, USA.
authors.forename: Zifan
authors.initials: Z
authors.lastname: Gong
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD, 20742, USA.
authors.forename: Mackenzie T
authors.initials: MT
authors.lastname: Walls
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD, 20742, USA.
authors.forename: Alisha N
authors.initials: AN
authors.lastname: Karley
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD, 20742, USA. ajkarl@umd.edu.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Karlsson
mesh_terms: D002176:Candida albicans / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C437783:pVEC peptide; D049452:Green Fluorescent Proteins
keywords: Candida albicans; Cell-penetrating peptide; Cellular uptake; Glycine–serine linker; Protein fusion; Recombinant protein expression