kth.sePublikationer
Ändra sökning
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0002-4751-2519
KTH, Skolan för bioteknologi (BIO), Proteinteknologi.ORCID-id: 0000-0003-0605-8417
2015 (Engelska)Ingår i: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, nr 4, s. 564-574Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2015. Vol. 10, nr 4, s. 564-574
Nyckelord [en]
Antibody labeling, Biomolecular engineering, Protein G, Site-specific biotinylation, Unnatural amino acid
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:kth:diva-164259DOI: 10.1002/biot.201400808ISI: 000352636500008PubMedID: 25655274Scopus ID: 2-s2.0-84964199667OAI: oai:DiVA.org:kth-164259DiVA, id: diva2:805036
Forskningsfinansiär
Vetenskapsrådet
Anmärkning

QC 20150427

Tillgänglig från: 2015-04-14 Skapad: 2015-04-14 Senast uppdaterad: 2022-06-23Bibliografiskt granskad
Ingår i avhandling
1. Engineering of small IgG binding domains for antibody labelling and purification
Öppna denna publikation i ny flik eller fönster >>Engineering of small IgG binding domains for antibody labelling and purification
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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. 

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2016. s. 82
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:15
Nyckelord
Antibody, labelling, purification, Protein G, Protein A, protein engineering, protein design, combinatorial selection
Nationell ämneskategori
Teknik och teknologier Biokemi och molekylärbiologi
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-191303 (URN)978-91-7729-093-3 (ISBN)
Externt samarbete:
Disputation
2016-09-30, M2, Brinellvägen 64, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-09-09 Skapad: 2016-08-26 Senast uppdaterad: 2022-06-22Bibliografiskt granskad

Open Access i DiVA

Fulltext saknas i DiVA

Övriga länkar

Förlagets fulltextPubMedScopus

Person

Kanje, SaraHober, Sophia

Sök vidare i DiVA

Av författaren/redaktören
Kanje, SaraHober, Sophia
Av organisationen
Proteinteknologi
I samma tidskrift
Biotechnology Journal
Medicin och hälsovetenskap

Sök vidare utanför DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetricpoäng

doi
pubmed
urn-nbn
Totalt: 218 träffar
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf