title: Construction of signal peptide-FRET fusion expression vectors and their expressions in NIH3T3 cells.
abstract: OBJECTIVE
  To construct the fluorescence protein vector pairs with toll-like receptor 4 (TLR4) signal peptide, namely pECFP-C1-SP and pEYFP-C1-SP, therefore to make the application of fluorescence resonance energy transfer (FRET) possible in the study of the membrane protein interaction during lipopolysaccharide (LPS) recognition.
  
  
  METHODS
  pECFP-C1-SP and pEYFP-C1-SP were constructed by means of site-directed mutagenesis, and the constructed plasmids were transiently transfected into NIH3T3 cells via lipofectamin to observe their intracellular expressions under a fluorescence microscope.
  
  
  RESULTS
  DNA sequence analysis attested the validity of the constructed fluorescence vectors with signal peptide for FRET, and the expression of the vectors was located principally on the cell membrane as observed under fluorescence microscope.
  
  
  CONCLUSIONS
  The constructed vectors TLR4 signal peptide are valid and capable of expressing on the cell membrane, therefore they can be effectively used in the study of the interaction between the membrane proteins.
authors.affiliation: Key Laboratory for Shock and Microcirculation of PLA, First Military Medical University, Guangzhou 510515, China.
authors.forename: Xue-gang
authors.initials: XG
authors.lastname: Sun
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.forename: Jing-hua
authors.initials: JH
authors.lastname: Liu
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: An
authors.forename: Hong-le
authors.initials: HL
authors.lastname: Li
authors.forename: Fei-yue
authors.initials: FY
authors.lastname: Xing
authors.forename: Li-hua
authors.initials: LH
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
mesh_terms: D016475:3T3 Cells; D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
chemical_list: D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors