Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications

• For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Biosensors; Characterization; Cytology; Flow cytometry; Gene expression; Proteins; Reaction rates; Affibody molecules; Binding molecules; Cell surface; Immunoglobulins; Staphylococcal cell surface; Biochemical engineering; albumin; immunoglobulin G; bacterium; cell organelle; flow cytometry; protein; article; binding affinity; cell surface; combinatorial chemistry; display system; dissociation constant; equilibrium constant; flow cytometry; molecular cloning; molecular model; nonhuman; protein binding; protein domain; protein engineering; protein expression; protein purification; reliability; reproducibility; Staphylococcus; surface property; Antibodies; Bacterial Outer Membrane Proteins; Binding Sites; Biosensing Techniques; Flow Cytometry; Humans; Immunoglobulin G; Protein Engineering; Staphylococcus

Molecular biology

Molekylärbiologi

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)