Functional expression of anti-hepatitis B virus (HBV) preS2 antigen scFv by <Emphasis Type="Italic">cspA</Emphasis> promoter system in <Emphasis Type="Italic">Escherichia coli</Emphasis> and application as a recognition molecule for single-walled carbon nanotube (SWNT) field effect transistor (FET) |
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Authors: | Sun-Ae Jun Dong Hyun Nam Young-Seob Lo Ju-Jin Kim Hyunju Chang Jeong-O Lee Yong Hwan Kim Byoung-In Sang |
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Institution: | (1) Danish Technological Institute, Holbergsvej 10, 6000 Kolding, Denmark;(2) Laboratory of BioDesign, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark |
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Abstract: | The preS2 antigens of hepatitis B virus (HBV), which causes a serious health problem in the world, have been implicated in
hepatocyte cell binding and viral penetration. Therefore, the importance of antibody production against preS2 antigen for
early diagnosis of HBV has been well established. In this study, the recombinant HBV preS2 single chain variable fragment
(scFv) antibody was successfully expressed in E. coli with the novel cold shock vector (pCold) under the cspA promoter, and its expression level was compared with the pET vector under the T7 promoter. Additionally, a host with an oxidizing
cytoplasm, E. coli trxB/gor double mutant, was used to improve the soluble expression. The anti-HBV preS2 scFv using pCold vector was successfully expressed
in a soluble and functional form in both wild type and double mutant E. coli, while the scFv using the pET vector was expressed in an insoluble form in spite of using a double mutant providing an oxidizing
environment. The induction with 0.05 mM IPTG showed a 2-fold higher functional expression compared to induction with 1 mM
IPTG, and the functional expression at the induction temperature (15°C), which is optimal temperature for pCold vector, was
improved 2-fold and 3- fold at 4 and 25°C, respectively. The efficacy of anti-HBV preS2 scFv for detecting HBV preS2 antigen
was tested and verified by using Ni-decorated single-walled carbon nanotube (SWNT) field effect transistors. |
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