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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
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0002-5391-600X
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2000 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 296, no 1, 87-102 p.Article in journal (Refereed) Published
Abstract [en]

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 lentils 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-Pro- 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-Ala- L-Pro-Phe(P)-diphenylester ter 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.

Place, publisher, year, edition, pages
2000. Vol. 296, no 1, 87-102 p.
Keyword [en]
phage display, subtilisin, specificity, biotinylated inhibitors, enzyme selection, bacillus-lentus, intramolecular chaperone, escherichia-coli, mutational replacements, denatured subtilisin, assisted catalysis, directed evolution, crystal-structure, serine proteases, binding-proteins
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-13274DOI: 10.1006/jmbi.1999.3437ISI: 000085295900007OAI: diva2:323031
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2012-06-13Bibliographically approved
In thesis
1. Protein Engineering by Directed Evolution and Rational Design
Open this publication in new window or tab >>Protein Engineering by Directed Evolution and Rational Design
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2001. 87 p.
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
National Category
Industrial Biotechnology
urn:nbn:se:kth:diva-3171 (URN)91-7283-102-2 (ISBN)
Public defence
2001-06-01, 00:00
QC 20100609Available from: 2001-05-31 Created: 2001-05-31 Last updated: 2010-06-09Bibliographically approved

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