title: Discovery and characterization of a new cell-penetrating protein.
abstract: We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.
authors.affiliation: Artie McFerrin Department of Chemical Engineering, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Rudo L
authors.initials: RL
authors.lastname: Simeon
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Chamoun
authors.forename: Thomas
authors.initials: T
authors.lastname: McMillin
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011995:Recombination, Genetic; D055672:Static Electricity; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D057846:Cell-Penetrating Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
references.citation: J Neurosci Methods. 1990 Jan;31(1):43-6
references.pmid: 2308380
references.citation: Nucleic Acids Res. 1996 Nov 15;24(22):4592-3
references.pmid: 8948654
references.citation: J Neurosci. 2005 Oct 5;25(40):9152-61
references.pmid: 16207874
references.citation: J Gen Virol. 1996 Sep;77 ( Pt 9):2173-82
references.pmid: 8811017
references.citation: J Cell Biol. 1990 Dec;111(6 Pt 2):2931-8
references.pmid: 2148564
references.citation: Bioconjug Chem. 2002 Jan-Feb;13(1):3-6
references.pmid: 11792172
references.citation: Biochim Biophys Acta. 2012 Mar;1818(3):502-11
references.pmid: 22155257
references.citation: Adv Drug Deliv Rev. 2009 Sep 30;61(11):953-64
references.pmid: 19538995
references.citation: J Am Chem Soc. 2005 Sep 14;127(36):12492-3
references.pmid: 16144388
references.citation: J Biol Chem. 2004 Aug 6;279(32):33855-64
references.pmid: 15159406
references.citation: Biotechniques. 1991 Apr;10(4):520-5
references.pmid: 1867862
references.citation: Gene. 1992 Feb 15;111(2):229-33
references.pmid: 1347277
references.citation: Drug Metab Dispos. 2011 Oct;39(10):1946-53
references.pmid: 21712433
references.citation: Methods Mol Biol. 2011;683:553-72
references.pmid: 21053156
references.citation: Dev Cell. 2006 Jun;10(6):839-50
references.pmid: 16740485
references.citation: J Control Release. 2005 Dec 5;109(1-3):77-85
references.pmid: 16271792
references.citation: Science. 2006 May 19;312(5776):1027-30
references.pmid: 16709779
references.citation: Chem Soc Rev. 2011 Jul;40(7):3638-55
references.pmid: 21566806
references.citation: J Virol. 2000 Nov;74(22):10827-33
references.pmid: 11044132
references.citation: J Invest Dermatol. 2009 Mar;129(3):590-8
references.pmid: 18787533
references.citation: Methods. 2001 Jul;24(3):247-56
references.pmid: 11403574
references.citation: Mol Plant Microbe Interact. 2011 Jan;24(1):25-36
references.pmid: 21138375
references.citation: Cell. 1988 Dec 23;55(6):1189-93
references.pmid: 2849510
references.citation: Mol Ther. 2009 Jan;17(1):95-103
references.pmid: 18957965
references.citation: J Biol Chem. 2000 Oct 27;275(43):33213-21
references.pmid: 10913125
references.citation: FEBS Lett. 2000 Oct 6;482(3):265-8
references.pmid: 11024473
references.citation: Nat Biotechnol. 2001 Jul;19(7):640-4
references.pmid: 11433275
references.citation: Curr Protein Pept Sci. 2003 Apr;4(2):97-104
references.pmid: 12678849
references.citation: Angew Chem Int Ed Engl. 2004 Oct 4;43(39):5242-6
references.pmid: 15455428
references.citation: Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413-7
references.pmid: 2823261
references.citation: J Pept Res. 2000 Nov;56(5):318-25
references.pmid: 11095185
references.citation: Biochem J. 1989 Jul 15;261(2):389-93
references.pmid: 2505760
references.citation: Chem Commun (Camb). 2005 Feb 7;(5):571-7
references.pmid: 15672140
references.citation: Gene. 1996;173(1 Spec No):33-8
references.pmid: 8707053
references.citation: Cell Stem Cell. 2009 Jun 5;4(6):472-6
references.pmid: 19481515
references.citation: Nat Biotechnol. 2001 Dec;19(12):1173-6
references.pmid: 11731788
references.citation: J Cell Sci. 2001 Mar;114(Pt 5):837-8
references.pmid: 11181166
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7297-301
references.pmid: 7638184
references.citation: ACS Chem Biol. 2010 Aug 20;5(8):747-52
references.pmid: 20545362
references.citation: J Cell Sci. 1994 Dec;107 ( Pt 12):3461-8
references.pmid: 7706397
references.citation: Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):664-8
references.pmid: 8290579
references.citation: Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1864-8
references.pmid: 1672046
references.citation: Bioconjug Chem. 2004 Jul-Aug;15(4):831-40
references.pmid: 15264871
references.citation: ACS Nano. 2011 Feb 22;5(2):1385-94
references.pmid: 21268592
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9682-6
references.pmid: 8790391
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6111-6
references.pmid: 19307578
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Chem Biol. 2012 Jul 27;19(7):831-43
references.pmid: 22840771
references.citation: J Immunol. 1997 Sep 1;159(5):2462-7
references.pmid: 9278339
references.citation: Chem Biol. 2011 Jul 29;18(7):833-8
references.pmid: 21802004
references.citation: J Biol Chem. 1999 Dec 17;274(51):36267-73
references.pmid: 10593915
references.citation: Br J Pharmacol. 2009 May;157(2):195-206
references.pmid: 19309362
references.citation: Methods Enzymol. 1996;266:540-53
references.pmid: 8743705
references.citation: Biochim Biophys Acta. 2009 Mar;1790(3):198-207
references.pmid: 19402206