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Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.ORCID-id: 0000-0001-9423-0541
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.
KTH, Skolan för bioteknologi (BIO), Molekylär Bioteknologi.ORCID-id: 0000-0002-9282-0174
2007 (engelsk)Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, nr 21, s. 6714-6721Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

sted, utgiver, år, opplag, sider
2007. Vol. 73, nr 21, s. 6714-6721
Emneord [en]
Biosensors; Characterization; Cytology; Flow cytometry; Gene expression; Proteins; Reaction rates; Affibody molecules; Binding molecules; Cell surface; Immunoglobulins; Staphylococcal cell surface; Biochemical engineering; albumin; immunoglobulin G; bacterium; cell organelle; flow cytometry; protein; article; binding affinity; cell surface; combinatorial chemistry; display system; dissociation constant; equilibrium constant; flow cytometry; molecular cloning; molecular model; nonhuman; protein binding; protein domain; protein engineering; protein expression; protein purification; reliability; reproducibility; Staphylococcus; surface property; Antibodies; Bacterial Outer Membrane Proteins; Binding Sites; Biosensing Techniques; Flow Cytometry; Humans; Immunoglobulin G; Protein Engineering; Staphylococcus
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-7844DOI: 10.1128/AEM.01432-07ISI: 000250700600003PubMedID: 17873070Scopus ID: 2-s2.0-35948962788OAI: oai:DiVA.org:kth-7844DiVA, id: diva2:12985
Merknad
QC 20100809Tilgjengelig fra: 2007-12-14 Laget: 2007-12-14 Sist oppdatert: 2017-12-14bibliografisk kontrollert
Inngår i avhandling
1. Staphylococcal surface display for protein engineering and characterization
Åpne denne publikasjonen i ny fane eller vindu >>Staphylococcal surface display for protein engineering and characterization
2007 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KHT, 2007. s. x, 95
Emneord
affibody, combinatorial library, epitope mapping, Gram-positive bacteria, protein engineering, staphylococcal surface display
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-4584 (URN)978-91-7178-834-4 (ISBN)
Disputas
2008-01-11, FD5, Albanova, Roslagstullsbacken 21, Stockholm, 09:00
Opponent
Veileder
Merknad
QC 20100809Tilgjengelig fra: 2007-12-14 Laget: 2007-12-14 Sist oppdatert: 2010-08-09bibliografisk kontrollert

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