Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase A

Andersson, Ken G.KTH,Proteinteknologi,Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. (författare)

QC 20170904
Combinatorial protein engineering using libraries displayed on various microorganisms is a powerful method forgeneration of new affinity proteins. Successful efforts often result in broad panels of isolated binders, which are thentypically subcloned, produced, purified and characterized in various assays. Many such assays also require conjugation tofor example reporters or other functional molecules and the downstream production and modification thus tends to be verylaborious and limits the number of candidates that can be screened. Staphylococcal sortase A is a natural transpeptidasethat catalyzes the ligation between a LPXTG motif and N-terminal glycines and is today used in a variety of applicationsfor site-specific conjugation of different molecules to recombinant proteins. We have previously developed a surfacedisplay method for combinatorial protein engineering of Affibody molecules on the outer membrane of E. coli usingautodisplay. Here, we introduced a sortase-A recognition motif into the displayed recombinant proteins and evaluatedsortase-mediated release and specific conjugation of various reporters to Affibody molecules. The approach has potentialto significantly increase the flexibility and throughput of downstream characterization of affinity proteins after directedevolution using cell display and FACS.

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
AIDA-I
Affibody molecules
autodisplay
autotransporter
bioconjugation
combinatorial protein engineering
directed evolution
protein ligation
sortase A
Bioteknologi
Biotechnology

Sjöstrand, NannaKTH,Proteinteknologi,ivision of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.(Swepub:kth)u1uzpchx (författare)
Löfblom, JohnKTH,Proteinteknologi,Proteomik och nanobioteknologi,Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.(Swepub:kth)u1z7cgkl (författare)
KTHProteinteknologi (creator_code:org_t)

Av författaren/redakt...: Andersson, Ken G ...; Sjöstrand, Nanna; Löfblom, John

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