Search: onr:"swepub:oai:DiVA.org:kth-23352" >
Improving the toler...
Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach
-
- Linhult, M. (author)
- Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden
-
- Gulich, S. (author)
- Gülich, S., Dept. of Molec. Biophys./Biochem., Yale University, New Haven, CT, United States
-
- Gräslund, Torbjörn (author)
- KTH,Bioteknologi,Gräslund, T., Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden
-
show more...
-
- Simon, A. (author)
- Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden
-
- Karlsson, Martin (author)
- Linköpings universitet,Tekniska högskolan,Biokemi
-
- Sjoberg, A. (author)
- Sjöberg, A., Affibody AB, Bromma, Sweden
-
- Nord, K. (author)
- Affibody AB, Bromma, Sweden
-
- Hober, Sophia (author)
- KTH,Bioteknologi,Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden, Department of Biotechnology, KTH, Royal Institute of Technology, SE-106 91 Stockholm, Sweden
-
show less...
-
Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden Gülich, S, Dept. of Molec. Biophys./Biochem., Yale University, New Haven, CT, United States (creator_code:org_t)
- 2004-02-27
- 2004
- English.
-
In: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 55:2, s. 407-416
- Related links:
-
https://urn.kb.se/re...
-
show more...
-
https://doi.org/10.1...
-
https://urn.kb.se/re...
-
show less...
Abstract
Subject headings
Close
- Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its nonengineered form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.
Keyword
- affinity chromatography
- deamidation
- protein A
- purification
- stabilization
- Z domain
- bacterial receptor domain
- escherichia-coli
- binding domain
- staphylococcus-aureus
- secondary structure
- b-domain
- deamidation
- stability
- sequence
- affinity
- TECHNOLOGY
Publication and Content Type
- ref (subject category)
- art (subject category)
Find in a library
-
Proteins
(Search for host publication in LIBRIS)
To the university's database