Enabling Glycosyltransferase Evolution: A Facile Substrate‐Attachment Strategy for Phage‐Display Enzyme Evolution

Love, Kerry R.; Swoboda, Jonathan G.; Noren, Christopher J.; Walker, Suzanne

doi:10.1002/cbic.200600018

Cited by 29 publications

(18 citation statements)

References 32 publications

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“…In contrast to previous attempts that include unnatural or noncanonical amino acids in directed evolution experiments (21,22), unnatural amino acids in our system are genetically encoded and require only the unique codon, TAG, for site-specific incorporation. Therefore, translation with unnatural amino acids utilizes the same fundamental paradigm as translation with natural amino acids, resulting in unrestricted 21 aa protein evolution.…”

Section: Discussionmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

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

143

132

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We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V H CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded ''synthetic'' genetic code can confer a selective advantage in the directed evolution of proteins with specific properties. directed evolution ͉ phage display ͉ unnatural amino acids W ith few exceptions, the genetic codes of all known organisms specify only the 20 canonical amino acids for protein synthesis. Yet it is quite possible that the ability to encode additional amino acids and their corresponding chemical functionalities would be evolutionarily advantageous, especially since nature's choice of 20 could have been arbitrarily fixed at the point of transition between communal and Darwinian evolution paradigms and subsequently sustained by the code's inertia (1). Furthermore, in the limited scope of laboratory-directed evolution, which concerns only one or few specific functions over a short time rather than general organismal fitness over thousands of years, one can easily envision a selective advantage conferred by additional amino acids. Recent developments in our laboratory allow us to explore this possibility. Specifically, orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs capable of incorporating various unnatural amino acids into proteins in response to unique nonsense and frameshift codons have been added to the translational machinery of Escherichia coli (2). These E. coli (X-E. coli) can now be used for evolution of protein function wherein 21 building blocks, rather than the common 20, are available.Several unnatural amino acids were initially chosen, on the basis of their unique chemistries, for use in our system. For example, X-E. coli genetically encoding the bidentate metalchelating amino acid bipyridyl-alanine (3) are well-suited for the evolution of redox and hydrolytic catalysts, as metal ion binding would not require preorganized primary and secondary ligand shells. Similarly, X-E. coli encoding the reactive 4-boronophenylalanine (4) are well-suited for evolution of receptors specific for glycoproteins or serine protease inhibitors, because the boronate group can form covalent complexes with diols or reactive serine residues. In addition, X-E. coli genetically encoding otherwise posttranslationally modified amino acids, such as sulfotyrosine (5), can be used for evolution of properties that exploit the unique chemical characterist...

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Section: Discussionmentioning

confidence: 99%

Protein evolution with an expanded genetic code

Liu

Mack

Tsao

et al. 2008

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

143

132

View full text Add to dashboard Cite

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“…Thus, the development of high-throughput assays for screening enzyme activities is a critical component of protein engineering efforts. [210,[302][303][304] Recently, the directed evolution of CstII, a sialyltransferase of the GT-A family, [303] and OleD, a macrolide resistance glucosyltrasferase of the GT-B family (that catalyzes 282! 283, Scheme 26 b), [210] have been reported.…”

Section: Protein Engineering Of Glycosyltransferasesmentioning

confidence: 99%

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

Thibodeaux

Melançon

Liu³

2008

Angew Chem Int Ed

390

397

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Many biologically active small-molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and alter the glycosylation patterns of natural products through metabolic pathway engineering and enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity.

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“…Development of other screening methods such as the use of a pH indicator [201] and phage display [202] should also facilitate the detection of promiscuous and powerful GTs via directed evolution.…”

Section: Scheme 18mentioning

confidence: 99%