A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry.
Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host ( approximately 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
Kronqvist
Nina
N
Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm.
Löfblom
John
J
Jonsson
Andreas
A
Wernérus
Henrik
H
Ståhl
Stefan
S
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002942:Circular Dichroism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013210:Staphylococcus / Q000235:genetics* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014409:Tumor Necrosis Factor-alpha