Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0002-9282-0174
Show others and affiliations
2007 (English)In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 102, no 3, 736-747 p.Article in journal (Refereed) Published
Abstract [en]

The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries.

Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population.

Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation.

Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.

Place, publisher, year, edition, pages
2007. Vol. 102, no 3, 736-747 p.
Keyword [en]
affibody; cell surface display; electroporation; Gram positive; Staphylococcus carnosus; transformation
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-7845DOI: 10.1111/j.1365-2672.2006.03127.xISI: 000244243900015Scopus ID: 2-s2.0-33847018452OAI: oai:DiVA.org:kth-7845DiVA: diva2:12986
Note
QC 20100726Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Staphylococcal surface display for protein engineering and characterization
Open this publication in new window or tab >>Staphylococcal surface display for protein engineering and characterization
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution.

Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering.

In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins.

Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization.

Place, publisher, year, edition, pages
Stockholm: KHT, 2007. x, 95 p.
Keyword
affibody, combinatorial library, epitope mapping, Gram-positive bacteria, protein engineering, staphylococcal surface display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4584 (URN)978-91-7178-834-4 (ISBN)
Public defence
2008-01-11, FD5, Albanova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Supervisors
Note
QC 20100809Available from: 2007-12-14 Created: 2007-12-14 Last updated: 2010-08-09Bibliographically approved
2. Staphylococcal surface display in directed evolution
Open this publication in new window or tab >>Staphylococcal surface display in directed evolution
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. x, 78 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:16
Keyword
affibody, combinatorial library, directed evoluation, Gram-positive bacteria. protein engineering
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11555 (URN)978-91-7415-418-4 (ISBN)
Public defence
2009-11-27, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:15 (English)
Opponent
Supervisors
Note

QC 20100726

Available from: 2009-11-20 Created: 2009-11-20 Last updated: 2012-12-14Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textScopusWiley Online Library

Authority records BETA

Löfblom, JohnUhlén, MathiasStåhl, Stefan

Search in DiVA

By author/editor
Löfblom, JohnKronqvist, NinaUhlén, MathiasStåhl, StefanWernérus, Henrik
By organisation
Molecular Biotechnology
In the same journal
Journal of Applied Microbiology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 185 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf