논문 (학술지)
Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors
| 등록번호 | RPMS-2018-0190682424 | SCI 구분
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※구분 : SCI(SCIE포함), 비SCI |
SCI |
|---|---|---|---|
| 저자명 (주·공동저자) | Bong Ji-Hong; Song Hyun-Woo; Kim Tae-Hun; Kang Min-Jung; Jose Joachim; Pyun Jae-Chul | ||
| 논문구분 | 국외전문학술지 | 학술지명 | BIOSENSORS & BIOELECTRONICS |
| ISSN | 0956-5663 | 학술지 출판일자 | 2018-01-01 |
| 학술지 볼륨번호 | 102 | 논문페이지 | 600 ~ 609 |
| 학술지 임팩트팩터 | 0.0 | 기여율 | 25 % |
| DOI | 10.1016/j.bios.2017.12.009 | ||
| 초록 | Abstract In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Highlights Anti-NEF scFv was autodisplayed on the outer membrane (OM) of Escherichia coli . Autodisplayed anti-NEF scFv was oxidized to form disulfide bonds by using glutathione. The activity of scFv was analyzed by FACS-based immunoassay and SPR biosensor. Mutants were made to confirm the formation of disulfides after the reaction of glutation. | ||
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