Linked Life Data

20716446

Source:http://linkedlifedata.com/resource/pubmed/id/20716446

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2010-10-11
pubmed:abstractText
Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1096-0279
pubmed:author
pubmed:copyrightInfo
Copyright © 2010 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:volume
74
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
162-8
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: using structural genomics tools for a challenging target.
pubmed:affiliation
AgResearch Structural Biology Laboratory, School of Biological Sciences, University of Auckland, Auckland, New Zealand. t.lee@auckland.ac.nz
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't