Cited 0 time in webofscience Cited 0 time in scopus

Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point

Title
Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point
Authors
Jeon, WB[Jeon, Won Bae]
DGIST Authors
Jeon, WB[Jeon, Won Bae]
Issue Date
2010-05-31
Citation
BMB Reports, 43(5), 319-324
Type
Article
Article Type
Article
Keywords
AnimalAnimalsChemistryCysteineCysteine ContentEndopeptidasesEscherichia ColiEscherichia Coli ProteinEscherichia Coli ProteinsEukaryotaExperimental BioinformaticsGeneticsHigh Throughput PurificationHumanHumansHybrid ProteinIsoelectric PointIsolation and PurificationMaltose-Binding ProteinMaltose Binding ProteinMolecular WeightPeriplasmic Binding ProteinPeriplasmic Binding ProteinsPlant ProteinsProteinaseRecombinant Fusion ProteinsRetrospective StudiesRetrospective StudyStructural GenomicsTEV ProteaseTobacco Etch VirusVegetable Protein
ISSN
1976-6696
Abstract
Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pl) of the target increased. This pattern of pl versus overall success rate strongly suggests that pl should be incorporated into target scoring criteria with a threshold value. [BMB reports 2010; 43(5): 319-324]
URI
http://hdl.handle.net/20.500.11750/3521
DOI
10.5483/BMBRep.2010.43.5.319
Publisher
Korean Society for Molecular and Cellular Biology
Related Researcher
Files:
Collection:
Companion Diagnostics and Medical Technology Research Group1. Journal Articles


qrcode mendeley

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE