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Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-4751-2519
KTH, School of Biotechnology (BIO), Protein Technology.
Karolinska institutet, Stockholm.
Karolinska institutet, Stockholm.
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2016 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812Article in journal (Refereed) Published
Abstract [en]

Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody’s binding to its target. Moreover, an emerging field for therapeutics is antibody–drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-191010DOI: 10.1021/acs.bioconjchem.6b00346OAI: oai:DiVA.org:kth-191010DiVA: diva2:954334
Note

QC 20160822

Available from: 2016-08-22 Created: 2016-08-22 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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:15
Keyword
Antibody, labelling, purification, Protein G, Protein A, protein engineering, protein design, combinatorial selection
National Category
Engineering and Technology Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
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)
Opponent
Supervisors
Available from: 2016-09-09 Created: 2016-08-26 Last updated: 2016-09-09Bibliographically approved

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Kanje, Saravon Witting, EmmaHober, Sophia
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