Background Sodium taurocholate co -transportering polypeptide (NTCP, SLC10A1 ) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide. Methods Recombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants. Results Sub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell. Conclusion Dimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell. General significance Our results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.
Yunhua Zhang,Jinyun Ji,Xiao Zhao,Ruoheng Jin,Jianhong Li,Hu Wan
A previous study demonstrated that a β- N -acetyl- d -hexosaminidase ( NlHex4 ) from the brown planthopper could serve as a new potential target for biopesticides due to its requirement for molting, and lethal phenotypes were observed via RNA interference . However, the enzymatic properties of NlHex4 remain unclear. In the present study, NlHex4 was expressed, purified and characterized. Recombinant NlHex4 was functionally expressed in baculovirus-infected insect cells and purified with a molecular weight of approximately 70 kDa. Recombinant NlHex4 had an optimal pH and temperature of 6.0 and 60 °C, respectively. Moreover, recombinant NlHex4 exhibited thermal stability at 50 °C for at least 1 h. Furthermore, the activity of recombinant NlHex4 was inhibited by the metal ions Fe 2 + and Fe 3 + and the serine protease inhibitor phenylmethanesulfonyl fluoride. These results reveal the enzymatic properties of NlHex4 and provide a basis for future investigation of the interaction between HEX and chitin oligosaccharide .
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