Change search
ReferencesLink to record
Permanent link

Direct link
In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-4751-2519
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0003-0605-8417
2015 (English)In: Biotechnology Journal, ISSN 1860-6768, Vol. 10, no 4, 564-574 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

Place, publisher, year, edition, pages
2015. Vol. 10, no 4, 564-574 p.
Keyword [en]
Antibody labeling, Biomolecular engineering, Protein G, Site-specific biotinylation, Unnatural amino acid
National Category
Medical and Health Sciences
URN: urn:nbn:se:kth:diva-164259DOI: 10.1002/biot.201400808ISI: 000352636500008PubMedID: 25655274ScopusID: 2-s2.0-84964199667OAI: diva2:805036
Swedish Research Council

QC 20150427

Available from: 2015-04-14 Created: 2015-04-14 Last updated: 2016-08-29Bibliographically approved
In thesis
1. Engineering of small IgG binding domains for antibody labelling and purification
Open this publication in new window or tab >>Engineering of small IgG binding domains for antibody labelling and purification
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In protein engineering, rational design and selection from combinatorial libraries are methods used to develop proteins with new or improved features. A very important protein for the biological sciences is the antibody that is used as a detecting agent in numerous laboratory assays. Antibodies used for these purposes are often ”man-made”, by immunising animals with the desired target, or by selections from combinatorial libraries. Naturally, antibodies are part of the immune defence protecting us from foreign attacks from e.g. bacteria or viruses. Some bacteria have evolved surface proteins that can bind to proteins abundant in the blood, like antibodies and serum albumin. By doing so, the bacteria can cover themselves in the host’s own proteins and through that evade being detected by the immune system. Two such proteins are Protein A from Staphylococcus aureus and Protein G from group C and G Streptococci. Both these proteins contain domains that bind to antibodies, one of which is denoted C2 (from Protein G) and another B (from Protein A). The B domain have been further engineered to the Z domain.

In this thesis protein engineering has been used to develop variants of the C2 and Z domains for site-specific labelling of antibodies and for antibody purification with mild elution. By taking advantage of the domains’ inherent affinity for antibodies, engineering and design of certain amino acids or protein motifs of the domains have resulted in proteins with new properties. A photo crosslinking amino acid, p-benzoylphenylalanine, have been introduced at different positions to the C2 domain, rendering three new protein domains that can be used for site-specific labelling of antibodies at the Fc or Fab fragment. These domains were used for labelling antibodies with lanthanides and used for detection in a multiplex immunoassay. Moreover, a library of calcium-binding loops was grafted onto the Z domain and used for selection of a domain that binds antibodies in a calcium dependent manner. This engineered protein domain can be used for the purification of antibodies using milder elution conditions, by calcium removal, as compared to traditional antibody purification. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 82 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:15
Antibody, labelling, purification, Protein G, Protein A, protein engineering, protein design, combinatorial selection
National Category
Engineering and Technology Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-191303 (URN)978-91-7729-093-3 (ISBN)
External cooperation:
Public defence
2016-09-30, M2, Brinellvägen 64, Stockholm, 10:00 (English)
Available from: 2016-09-09 Created: 2016-08-26 Last updated: 2016-09-09Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedScopus

Search in DiVA

By author/editor
Kanje, SaraHober, Sophia
By organisation
Protein Technology
In the same journal
Biotechnology Journal
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 61 hits
ReferencesLink to record
Permanent link

Direct link