Linked Life Data

10656819

Source:http://linkedlifedata.com/resource/pubmed/id/10656819

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2000-3-14
pubmed:abstractText
Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0022-2836
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
296
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
87-102
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:10656819-Alkanesulfonic Acids, pubmed-meshheading:10656819-Amino Acid Sequence, pubmed-meshheading:10656819-Bacillus, pubmed-meshheading:10656819-Binding Sites, pubmed-meshheading:10656819-Biotinylation, pubmed-meshheading:10656819-Catalysis, pubmed-meshheading:10656819-Cloning, Molecular, pubmed-meshheading:10656819-Directed Molecular Evolution, pubmed-meshheading:10656819-Drug Evaluation, Preclinical, pubmed-meshheading:10656819-Enzyme Activation, pubmed-meshheading:10656819-Enzyme Precursors, pubmed-meshheading:10656819-Genetic Variation, pubmed-meshheading:10656819-Lysine, pubmed-meshheading:10656819-Mutation, pubmed-meshheading:10656819-Peptide Library, pubmed-meshheading:10656819-Protease Inhibitors, pubmed-meshheading:10656819-Protein Processing, Post-Translational, pubmed-meshheading:10656819-Random Allocation, pubmed-meshheading:10656819-Recombinant Proteins, pubmed-meshheading:10656819-Serine Endopeptidases, pubmed-meshheading:10656819-Static Electricity, pubmed-meshheading:10656819-Streptavidin, pubmed-meshheading:10656819-Substrate Specificity
pubmed:year
2000
pubmed:articleTitle
Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors.
pubmed:affiliation
Laboratoire de Biochimie Physique et des biopolymères, Université catholique de Louvain, Place L. Pasteur, 1-1b, Louvain-la-Neuve, 1348, Belgium.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't