Selection of TNF-α binding affibody molecules using a β-lactamase protein fragment complementation assay

Abstract

Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naïve 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the beta-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PCA is a promising methodology for stringent selection of binders from complex naïve libraries to yield high affinity reagents with selective target binding characteristics.

Affinity maturation of a TNFα‐binding Affibody molecule by Darwinian survival selection

“…Although a variety of different read‐outs for PCA are possible, including FRET (Förster resonance energy transfer) [19], fluorescence [20] and luminescence [21], only reporter activities providing cell survival capability have been reported for selection trials. These include the use of mDHFR (mouse dihydrofolate reductase) as a reporter enzyme in both E. coli and yeast, where selection is based on an acquired resistance to trimethoprim [22], or the use of TEM‐1 β‐lactamase, where selection is based on resistance to Amp (ampicillin) [16,18].…”

“…We have described previously a β‐lactamase‐based PCA system [18] for the selection of Affibody molecules in which the target protein was co‐expressed in the periplasm of E. coli as C‐terminally fused to the first part (amino acids 26–197) of β‐lactamase via a 15‐residue‐long linker with library members N‐terminally fused via a similar linker to the latter part of the enzyme (amino acids 198–290) (Figure 1A). Both vector products were directed to the periplasm of E. coli by an OmpA signal peptide [23] and the inducible expression of both fusion proteins was under the control of a lac promoter.…”

“…Both vector products were directed to the periplasm of E. coli by an OmpA signal peptide [23] and the inducible expression of both fusion proteins was under the control of a lac promoter. Using a naïve Affibody molecule library of 10 9 complexity, several TNFα (tumour necrosis factor α)‐binding clones could be selected using a plate‐based survival selection methodology [18]. However, an alternative and attractive format for simple selection, possible for systems based on cell survival, would be to perform the selection in liquid medium by co‐culturing the entire library, relying on Darwinian principles of survival of the fittest as criteria for enrichment of desired variants by an expected capability for faster growth in an appropriate selective medium.…”

Design and evaluation of radiolabeled tracers for tumor imaging

“…A proof of concept was demonstrated by subjecting these first libraries, displayed on filamentous phage, to biopanning against three different targets, resulting in the isolation of micromolar‐affinity binders to all three targets. Since then, Affibody molecules toward more than 50 different targets have been generated, not only by phage display, but also by employing other selection systems such as cell surface display , ribosome display , and a protein fragment complementation assay . The complexity and size of the Affibody libraries has also increased considerably over the years, and Grimm et al recently reported on the construction of a naive ribosome display library with a library size of 10 11 Affibody molecules.…”

Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

“…From libraries constructed by combinatorial protein engineering principles, affibody molecules have been isolated based on their capability of selective binding to desired target proteins for later use in a variety of different affinity technology applications, including, for example, bioseparation, protein microarrays, in vivo imaging, and therapy [5–8]. The technologies used for linking genotype and phenotype during these selections have been phage display, cell display, and protein fragment complementation assay (PCA), or combinations thereof, all methods relying on the transformation of bacterial cells with plasmid DNA as one step in the experimental procedure, in practice limiting the sizes of libraries obtained [5, 9–11]. …”