Ribosome display of N-linked glycoproteins in cell-free extracts

Ribosome display is a powerful in vitro method for the selection and directed evolution of proteins expressed from combinatorial libraries. However, because ribosome display is typically performed with standard in vitro translation reagents, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this technological gap, here we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glyco-structural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free synthesis of N-glycoproteins. This integration yielded a novel capability for translating and displaying target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the encoding mRNAs remained stably attached to stalled ribosomes both before and after biopanning, thereby providing the genotype- glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-linked glycoproteins with advantageous biological and biophysical properties.