대장균에서 발현된 exoinulinase와 endoinulinase의 이차원적 전기영동과 MALDI-TOF MS를 이용한 동정

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.