Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries

Li, Yang; Gebhard, Mark C.; Li, Qing; Taft, Joseph M.; Georgiou, George; Iverson, Brent L.

doi:10.1073/pnas.1215994110

Cited by 121 publications

(187 citation statements)

References 37 publications

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“…Unlike this earlier report, however, here we demonstrate that SrtA may be further reprogrammed to possess fully altered, not merely broadened specificity. This capability is somewhat surprising given the mechanistic similarity between sortases and cysteine proteases (16) and the well-appreciated difficulty of the successful engineering of proteases with altered substrate specificities (32,33). We hypothesize a number of possible explanations for this success.…”

Section: Discussionmentioning

confidence: 99%

Reprogramming the specificity of sortase enzymes

Dorr

Ham

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

169

176

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Staphylococcus aureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophoreprotein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.protein evolution | enzyme specificity

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Section: Discussionmentioning

confidence: 99%

Reprogramming the specificity of sortase enzymes

Dorr

Ham

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

169

176

View full text Add to dashboard Cite

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“…We believe that this and similar knowledge may contribute to the redesign of MMPs to exhibit unconventional substrate specificity, which could have therapeutic, diagnostic, or other biotechnology applications (56,57). The substrate specificity of several proteases distinct from MMPs has been manipulated (58)(59)(60), but this was thought to be difficult to achieve with MMPs because of their presumed overlapping substrate specificity. MMPs not only offer a different set of potential target sequences but also have a longer substrate binding cleft than most serine proteases.…”

Section: Discussionmentioning

confidence: 99%

Basis for substrate recognition and distinction by matrix metalloproteinases

Ratnikov¹,

Cieplak²,

Gramatikoff³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for functionrelated predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k (obs) ] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.protease | specificity-determining positions | MMPs A paramount objective of biological research is to understand how sequence encodes function. Previously, functional regions in proteins were identified using large-scale mutagenesis (e.g., alanine scanning) (1). More recently, our insights are largely gained by computational approaches aimed at comparing sequences and structures of large protein sets across multiple genomes. The vast increase in the number of available sequences makes it possible to compare homology between sequences from genome projects to proteins of known structure and function and, as a result, identify functional similarities in silico (2-4). Because the global fold of most ordered proteins can be reliably predicted (5, 6) and because the catalytic residues of most classes of enzymes are either known or can be inferred (7,8), protein sequences can now be directly used to elucidate and classify major protein functions (9-12), and are even being extended to predict enzyme substrates (13).However, such classifications fail to explain the specialization and expansion of function that is required for organismal plasticity, complexity, and adaptability, all of which are normally driven by gene duplications and subsequent divergence. Computational approaches aimed at identifying functional distinctions across protein families have pri...

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“…Using this system, several proteins, including xylose isomerase (Ota et al, 2013), laccase (Nakanishi et al, 2012), and expansin-like protein , have been displayed on the yeast cell surface. Although numerous technologies for surface display systems have been reported of late (Fushimi et al, 2013;Qiu et al, 2014;Wilde et al, 2012;Yi et al, 2013;Zhang et al, 2013), the development of a wider variety of display systems would broaden the potential applications of surface engineered microorganisms toward industrial applications.…”

Section: Discussionmentioning

confidence: 98%