About: High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris

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About: High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

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type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
title	High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris
Creator	Yu, Xiao-Qiang Jin, Feng-Liang Ren, Shun-Xiang Xu, Xiao-Xia
source	Elsevier; Medline; PMC
abstract	Abstract Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Muts strain was much higher than that by Mut+ strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210mgl−1. With the (His)6-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin–magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin–magainin fusion protein and cleave it at the carboxyl terminus of ubquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.
has issue date	2009-06-30(xsd:dateTime)
bibo:doi	10.1016/j.pep.2008.08.010
bibo:pmid	18817875
has license	els-covid
sha1sum (hex)	e7447d3d1c60fbae9063f9f6d931c07ac43151d0
schema:url	https://doi.org/10.1016/j.pep.2008.08.010
resource representing a document's title	High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris
has PubMed Central identifier	PMC7128749
has PubMed identifier	18817875
schema:publication	Protein Expression and Purification
resource representing a document's body	covid:e7447d3d1c60fbae9063f9f6d931c07ac43151d0#body_text
is schema:about of	named entity 'DROSOPHILA MELANOGASTER' named entity 'SDS-PAGE' named entity 'sodium' named entity 'hydrolase' named entity 'expression' named entity 'RECOMBINANT' named entity 'AOX1' named entity 'HYDROLASE' named entity 'AUTHORS' named entity 'CARBOXYL' named entity 'THESE' named entity 'REN' named entity 'ALCOHOL' named entity 'LEVEL' named entity 'ISOPROPYL' named entity 'UBIQUITIN CARBOXYL-TERMINAL HYDROLASE' named entity 'HIGH' named entity 'ACTIVE' named entity 'EXPRESSION' named entity 'TERMINAL' named entity 'D. MELANOGASTER' named entity 'PICHIA PASTORIS' named entity 'THIOGALACTOSIDE' named entity 'USED' named entity 'AFFINITY CHROMATOGRAPHY' named entity 'SDS-PAGE' named entity 'ABBREVIATIONS' named entity 'METAL ION' named entity 'WORK' named entity 'IMAC' named entity 'UCH' named entity 'IPTG' named entity 'UBIQUITIN' named entity 'IMAC' named entity 'hydrolase' named entity 'carboxyl-terminal' named entity 'Abbreviations' named entity 'D. melanogaster' named entity 'ubiquitin' named entity 'sodium dodecyl sulfate' named entity 'alcohol' named entity 'carboxyl-terminal' named entity 'metal ion' named entity 'SDS-PAGE' named entity 'hydrolase' named entity 'ubiquitin' named entity 'IMAC' named entity 'magainin' named entity 'Bio-Rad' named entity 'fusion proteins' named entity 'magainin' named entity 'ubiquitin' named entity 'pH 8' named entity 'proteases' named entity 'culture medium' named entity 'gradient' named entity 'chelating' named entity '3CL protease' named entity 'ubiquitin' named entity 'methylotrophic' named entity 'Ni-NTA' named entity 'polyubiquitin chains' named entity 'ubiquitin' named entity 'chromatography' named entity 'magainin' named entity 'protein' named entity 'protein' named entity 'secretion' named entity 'thrombin' named entity 'phenotypes' named entity 'MMH' named entity 'affinity chromatography' named entity '1 ml' named entity 'Tricine' named entity 'Ni-NTA' named entity 'chelating' named entity 'GFP' named entity 'restriction site'

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