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Engineering of small IgG binding domains for antibody labelling and purification
KTH, School of Biotechnology (BIO), Protein Technology. (Sophia Hober)ORCID iD: 0000-0002-4751-2519
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 [en]
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: urn:nbn:se:kth:diva-191303ISBN: 978-91-7729-093-3OAI: oai:DiVA.org:kth-191303DiVA: diva2:956047
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
List of papers
1. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
Open this publication in new window or tab >>In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
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.

Keyword
Antibody labeling, Biomolecular engineering, Protein G, Site-specific biotinylation, Unnatural amino acid
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-164259 (URN)10.1002/biot.201400808 (DOI)000352636500008 ()25655274 (PubMedID)2-s2.0-84964199667 (ScopusID)
Funder
Swedish Research Council
Note

QC 20150427

Available from: 2015-04-14 Created: 2015-04-14 Last updated: 2016-08-29Bibliographically approved
2. Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain
Open this publication in new window or tab >>Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain
Show others...
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:nbn:se:kth:diva-191010 (URN)10.1021/acs.bioconjchem.6b00346 (DOI)
External cooperation:
Note

QC 20160822

Available from: 2016-08-22 Created: 2016-08-22 Last updated: 2016-08-29Bibliographically approved
3. Next generation of labeling reagents for quantitative and multiplexing immunoassays by use of LA-ICP-MS
Open this publication in new window or tab >>Next generation of labeling reagents for quantitative and multiplexing immunoassays by use of LA-ICP-MS
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Abstract. Immuno imaging by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a growing research field in the life sciences as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by use of LA-ICP-MS quantification of analytes is limited due to a non-controllable antibody labeling chemistry. in the presented proof-of-principle A novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine in the C-terminus of the protein. The modified C2 domain photo crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassays analyzed by use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody-antigen complexes in a multiplex Western Blot immunoassay was determined by LA-ICP-MS. 

National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-191160 (URN)
External cooperation:
Available from: 2016-08-24 Created: 2016-08-24 Last updated: 2016-08-29Bibliographically approved
4. An engineered Protein A derived domain for calcium dependent elution in antibody purification
Open this publication in new window or tab >>An engineered Protein A derived domain for calcium dependent elution in antibody purification
(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-191280 (URN)
External cooperation:
Note

QC 20160912

Available from: 2016-08-26 Created: 2016-08-26 Last updated: 2016-09-12Bibliographically approved

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