دورية أكاديمية
أعرض في EDS

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

التفاصيل البيبلوغرافية
العنوان: Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.
المؤلفون: Haggerty K; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Cantlay S; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Young E; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Cashbaugh MK; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Delatore Iii EF; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Schreiber R; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Hess H; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Komlosi DR; Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA., Butler S; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Bolon D; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Evangelista T; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Hager T; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Kelly C; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Phillips K; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Voellinger J; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA., Shanks RMQ; Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA., Horzempa J; Department of Biomedical Sciences, West Liberty University, West Liberty, WV, USA. Electronic address: joseph.horzempa@westliberty.edu.
المصدر: Molecular and cellular probes [Mol Cell Probes] 2024 Apr; Vol. 74, pp. 101956. Date of Electronic Publication: 2024 Mar 19.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Academic Press Country of Publication: England NLM ID: 8709751 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-1194 (Electronic) Linking ISSN: 08908508 NLM ISO Abbreviation: Mol Cell Probes Subsets: MEDLINE
أسماء مطبوعة: Original Publication: London ; New York : Academic Press, c1987-
مواضيع طبية MeSH: Francisella tularensis*/genetics , Francisella tularensis*/chemistry , Francisella tularensis*/metabolism, Lysine/metabolism ; Peptides/genetics ; Codon/genetics ; Protein Sorting Signals/genetics
مستخلص: Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFP Ft that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.
(Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)
References: Appl Environ Microbiol. 2011 Mar;77(5):1608-18. (PMID: 21193666)
Infect Immun. 2008 Dec;76(12):5843-52. (PMID: 18852251)
J Bacteriol. 2004 Jan;186(2):278-86. (PMID: 14702295)
PLoS One. 2010 Apr 01;5(4):e9952. (PMID: 20376351)
J Bacteriol. 2006 Jun;188(12):4244-52. (PMID: 16740931)
Microb Cell Fact. 2020 May 24;19(1):112. (PMID: 32448275)
Infect Immun. 2008 Jul;76(7):2833-42. (PMID: 18426871)
J Infect Dis. 2011 Jul 1;204(1):51-9. (PMID: 21628658)
Appl Environ Microbiol. 2008 Apr;74(7):2161-70. (PMID: 18245238)
BMC Microbiol. 2006 Jul 31;6:69. (PMID: 16879747)
Eur J Immunol. 2011 Apr;41(4):974-80. (PMID: 21442618)
Appl Microbiol Biotechnol. 2020 Mar;104(6):2411-2425. (PMID: 31993706)
Bio Protoc. 2017 Mar 20;7(6):. (PMID: 28660233)
J Bacteriol. 2022 Oct 18;204(10):e0026822. (PMID: 36121290)
Nucleic Acids Res. 2015 Mar 31;43(6):3022-32. (PMID: 25765653)
Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W70-4. (PMID: 18424795)
Infect Immun. 2002 Jun;70(6):2837-45. (PMID: 12010970)
Nat Methods. 2005 Dec;2(12):905-9. (PMID: 16299475)
Bioengineered. 2015;6(2):82-8. (PMID: 25617059)
Front Microbiol. 2012 Aug 09;3:226. (PMID: 22905031)
J Biosci Bioeng. 2017 May;123(5):540-546. (PMID: 28052816)
J Biol Chem. 2023 May;299(5):104676. (PMID: 37028767)
Nat Rev Immunol. 2010 May;10(5):353-64. (PMID: 20395980)
Nucleic Acids Res. 2022 Jan 7;50(D1):D439-D444. (PMID: 34791371)
Science. 2006 Apr 14;312(5771):217-24. (PMID: 16614209)
Front Cell Infect Microbiol. 2014 Mar 13;4:35. (PMID: 24660164)
Vaccine. 2016 Sep 22;34(41):4969-4978. (PMID: 27577555)
Science. 1994 Feb 11;263(5148):802-5. (PMID: 8303295)
Sci Rep. 2019 Aug 6;9(1):11418. (PMID: 31388083)
Front Cell Infect Microbiol. 2018 May 15;8:154. (PMID: 29868510)
Int J Mol Sci. 2021 Jul 22;22(15):. (PMID: 34360611)
Scand J Infect Dis. 2007;39(10):880-9. (PMID: 17886125)
Nat Methods. 2018 Jan;15(1):47-51. (PMID: 29320486)
Nat Biotechnol. 2004 Dec;22(12):1567-72. (PMID: 15558047)
Biotech Histochem. 2010 Dec;85(6):379-87. (PMID: 20109099)
Biotechnol Rep (Amst). 2021 Dec 31;33:e00697. (PMID: 35036336)
Methods. 2001 Dec;25(4):402-8. (PMID: 11846609)
Sci Rep. 2018 May 3;8(1):7009. (PMID: 29725025)
J Bacteriol. 2020 Jun 25;202(14):. (PMID: 32366588)
J Biotechnol. 2023 Sep 20;375:40-48. (PMID: 37652168)
Gene. 1996;173(1 Spec No):47-52. (PMID: 8707055)
Nat Commun. 2022 Aug 8;13(1):4614. (PMID: 35941164)
J Biol. 2010;9(2):12. (PMID: 20236488)
معلومات مُعتمدة: P20 GM103434 United States GM NIGMS NIH HHS; P30 EY008098 United States EY NEI NIH HHS; R01 EY032517 United States EY NEI NIH HHS; R15 HL147135 United States HL NHLBI NIH HHS
المشرفين على المادة: K3Z4F929H6 (Lysine)
0 (Peptides)
0 (Codon)
0 (Protein Sorting Signals)
تواريخ الأحداث: Date Created: 20240316 Date Completed: 20240401 Latest Revision: 20240504
رمز التحديث: 20240504
مُعرف محوري في PubMed: PMC11000650
DOI: 10.1016/j.mcp.2024.101956
PMID: 38492609
قاعدة البيانات: MEDLINE
الوصف
تدمد:1096-1194
DOI:10.1016/j.mcp.2024.101956