title: Profiling Protease Specificity: Combining Yeast ER Sequestration Screening (YESS) with Next Generation Sequencing.
abstract: An enzyme engineering technology involving yeast endoplasmic reticulum (ER) sequestration screening (YESS) has been recently developed. Here, a new method is established, in which the YESS platform is combined with NextGen sequencing (NGS) to enable a comprehensive survey of protease specificity. In this approach, a combinatorial substrate library is targeted to the yeast ER and transported through the secretory pathway, interacting with any protease(s) residing in the ER. Multicolor FACS screening is used to isolate cells labeled with fluorophore-conjugated antibodies, followed by NGS to profile the cleaved substrates. The YESS-NGS method was successfully applied to profile the sequence specificity of the wild-type and an engineered variant of the tobacco etch mosaic virus protease. Proteolysis in the yeast secretory pathway was also mapped for the first time in vivo revealing a major cleavage pattern of Ali/Leu-X-Lys/Arg-Arg. Here Ali is any small aliphatic residue, but especially Leu. This pattern was verified to be due to the well-known endogenous protease Kex2 after comparison to a newly generated Kex2 knockout strain as well as cleavage of peptides with recombinant Kex2 in vitro. This information is particularly important for those using yeast display technology, as library members with Ali/Leu-X-Lys/Arg-Arg patterns are likely being removed from screens via Kex2 cleavage without the researcher's knowledge.
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: Li
authors.identifier: 0000-0002-7835-6985
authors.initials: L
authors.lastname: Yi
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: Kam Hon
authors.initials: KH
authors.lastname: Hoi
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Marek
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
authors.affiliation: Department of Chemistry, ‡Department of Biomedical Engineering, §Department of Chemical Engineering, and ∥Section of Molecular Genetics and Microbiology, University of Texas , Austin, Texas 78712, United States.
authors.forename: Brent L
authors.initials: BL
authors.lastname: Iverson
mesh_terms: D004721:Endoplasmic Reticulum / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D012441:Saccharomyces cerevisiae / Q000378:metabolism*