Construction of signal peptide-FRET fusion expression vectors and their expressions in NIH3T3 cells.
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.
Sun
Xue-gang
XG
Key Laboratory for Shock and Microcirculation of PLA, First Military Medical University, Guangzhou 510515, China.
Song
Ge
G
Liu
Jing-hua
JH
An
Hai-yan
HY
Li
Hong-le
HL
Xing
Fei-yue
FY
Zhang
Li-hua
LH
Jiang
Yong
Y
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
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