(104e) Turning Ribosome Display Outside In For Intracellular Protein Engineering

Ribosome display is a powerful in vitro selection tool to identify proteins with enhanced functional properties (i.e., affinity, specificity, thermal stability) from large combinatorial libraries. However, one limitation of ribosome display is that it only works in vitro, where efficient translation is challenging and where cellular factors (i.e., chaperones and isomerases) that might be required for efficient protein folding are absent. Additionally, in vivo verification is typically needed to ensure that any functional improvements discovered in vitro are reproducible inside host cells, where engineered proteins will eventually be manufactured. A final limitation of ribosome display and several other in vitro platforms (e.g., phage display) with respect to antibody engineering is the need for purification and immobilization of the target antigen. To address these shortcomings, we have engineered a modified ribosome display strategy using the SecM translation arrest mechanism that enables antibody engineering directly in the cytosol of living cells. This is advantageous because: (1) expression and solubility of ribosome displayed proteins are intrinsic selection parameters imposed by the assay; and (2) the ?biopanning' process occurs in the cytoplasm via biosynthesis of stable antibody-ribosome-mRNA-antigen (ARMA) complexes, thereby obviating the need for antigen purification and immobilization. We will discuss how our ribosome display system has enabled stability maturation of functional antibody fragments in the E. coli cytoplasm. Such intracellular antibodies that are solubly expressed and functional in the cytoplasm are known as intrabodies and have great potential in functional genomics/proteomics efforts and in molecular medicine.