Robert G. Kuimelis

Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA‐protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion™ process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA‐protein fusion to create an addressable protein microarray that self‐assembles via hybridization to surface‐bound DNA capture probes. The nucleic acid component not only directs the mRNA‐protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA‐protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub‐attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.