Abstract As a common protein modification, asparagine-linked ( N- linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation at naïve sites as a strategy for engineering proteins with advantageous properties is currently limited by our inability to construct large collections of glycoproteins for interrogating the structural and functional consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis (SSGM) in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. Moreover, the resulting neoglycoproteins can be readily subjected to available medium- to high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and a human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N- glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by the N- linked glycan in a manner that critically depended on the precise location of the modification. Importantly, we anticipate that our workflow for creating and characterizing large ensembles of neoglycoproteins should provide access to unexplored regions of glycoprotein structural space and to custom-made glycoproteins with desirable properties.

Shotgun scanning glycomutagenesis: a simple and efficient strategy for constructing and characterizing neoglycoproteins