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대장균에서 발현된 exoinulinase와 endoinulinase의 이차원적 전기영동과 MALDI-TOF MS를 이용한 동정
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- Authors
- Advisor
- 김수일
- Issue Date
- 2003
- Publisher
- 서울대학교 대학원
- Keywords
- two-dimensional electrophoresis ; Two-dimensional electrophoresis ; isoelectric focusing ; Isoelectric focusing ; image analysis ; Image analysis ; MALDI-TOF MS ; Maldi-tof ms ; peptide fingerprinting ; Peptide fingerprinting
- Description
- 학위논문(석사)--서울대학교 대학원 :농생명공학부,2003.
- Abstract
- Protein identification in proteomic analysis requires many sequential steps
including sample preparation, IEF, two dimensional electrophoresis (2-DE),
image analysis, in-gel digestion with trypsin, matrix assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and
searches of molecular weight in peptide-mass databases. Among these steps,
well resolved 2-DE gels are required for successful proteomic analysis.
Resolution in 2-DE depends on many proceeding steps including sample
preparation, isoelectric focusing (IEF) and SDS-PAGE. However, due to
variations in these steps 2-DE does not always result in reproducible resolutions.
Optimization of these procedures is a foremost important issue in successful
proteomic analysis.
In this thesis, the following procedures were optimized using bacterial
proteins from E. coli BL21; i) sample preparation, ii) optimized Vhr in IEF, iii)
12.5% SDS-PAGE with fresh running buffer and iv) silver staining. These
optimized procedures produced a well-resolved 2-DE gel. Image analysis by
ImageMaster program followed by the in-gel digestion with trypsin identified
unique peptide peaks in MALDI-TOF MS.
These optimized conditions were employed to identify an exo- and an endoinulinase
protein which were expressed in E. coli BL21 with the introduced expression vector. Those two proteins were detected in 2-DE. Further
identifications using peptide fingerprinting in MALDI-TOF analysis clearly
identified these two foreign proteins along with other vector-encoded proteins.
These results support that the optimized procedures in this thesis are well
suitable for analyzing bacterial proteins.
- Language
- Korean
- URI
- http://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000059675
https://hdl.handle.net/10371/68510
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