Directed Evolution Using Stabilized Bacterial Peptide Display

Navaratna, Tejas; Atangcho, Lydia; Mahajan, Mukesh; Vivekanandan, Subramanian; Case, Marshall; Min, Andrew; Tresnak, Daniel T.; Thurber, Greg M.

doi:10.1021/jacs.9b10716

Cited by 30 publications

(54 citation statements)

References 74 publications

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“…The versatility of the display format supported efficient investigation of each these schemes. Bioorthogonal CuAAC and SPAAC reactions were readily achieved on the yeast surface, in line with previous ncAA-mediated functionalization results from our group and others on yeast [62][63][114][115] and E. coli 36,53,[116][117] expect this approach to be transferrable to other binding protein scaffolds. 75,[118][119][120][121][122] We note that the careful introduction of additional antibody diversity, such as using multiple frameworks, 22,25,65 may enhance antibody library performance while still supporting rapid chemical diversification.…”

Section: Discussionsupporting

confidence: 84%

“…52,123 In this regard, the simple synthetic antibodies and characterization strategies elucidated in this work could facilitate sideby-side evaluations of multiple chemical modification strategies. Finally, while many routes to prepare chemically modified peptides in display formats are now available, 29,[35][36][124][125][126][127][128][129][130][131] very few analogous approaches for the chemical diversification of proteins have been described. 53,60 The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering "drug-like" protein leads in high throughput.…”

Section: Discussionmentioning

confidence: 99%

“…Several strategies for expanding the chemical repertoire of antibodies are feasible, [29][30][31][32] including posttranslational modification of antibody structures containing canonical amino acids 31,[33][34] and the introduction of genetically encoded noncanonical amino acids (ncAAs). 32,[35][36][37][38] Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody-drug conjugates (ADCs). [39][40][41][42][43][44][45][46] However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; [46][47][48][49][50][51][52] the result is modular addition of new chemistries that leave antibody binding function unchanged.…”

Section: Introductionmentioning

confidence: 99%

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Chemical Diversification of Simple Synthetic Antibodies

et al. 2021

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Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in the canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. Using yeast displayed antibodies, we explored the presentation of noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs) and evaluated the properties of the resulting constructs. To enable systematic characterization of ncAA incorporation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We constructed a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR-H3). Screens against a series of immunoglobulins from three species resulted in the isolation of antibodies exhibiting moderate affinities (double-to triple-digit nanomolar affinities) and, in several cases, single-species specificity. These findings confirmed that antibody specificity can be mediated by a single CDR. With this constrained diversity, we were able to utilize additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Apparent binding affinities of ncAA-substituted synthetic antibodies on the yeast surface revealed that ncAA incorporation is generally well tolerated. However, changes in binding affinities did occur upon substitution, and varied based on factors including ncAA side chain identity, location of ncAA incorporation, and the ncAA incorporation machinery used. We further investigated chemical modifications facilitated by ncAA installation. Multiple azide-containing ncAAs supported both copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without abrogation of binding function following the installation of bulky probes. Similarly, several alkyne substitutions facilitated CuAAC without apparent disruption of binding function. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent linkages that could not be displaced by excess soluble antigen, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. These consistent behaviors between the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. More specifically, o...

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chemical Diversification of Simple Synthetic Antibodies

et al. 2021

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“…Navaratna et al . reported the stabilization of peptide evolution by E. coli displays 8 . Peptide stabilization was performed by click chemistry using bis-alkyne molecules, and stabilized peptides showed 4–9 times higher affinity and high protease stability.…”

Section: Introductionmentioning

confidence: 99%

Machine learning screening of bile acid-binding peptides in a peptide database derived from food proteins

Imai

Shimizu

Honda

2021

Sci Rep

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Bioactive peptides (BPs) are protein fragments that exhibit a wide variety of physicochemical properties, such as basic, acidic, hydrophobic, and hydrophilic properties; thus, they have the potential to interact with a variety of biomolecules, whereas neither carbohydrates nor fatty acids have such diverse properties. Therefore, BP is considered to be a new generation of biologically active regulators. Recently, some BPs that have shown positive benefits in humans have been screened from edible proteins. In the present study, a new BP screening method was developed using BIOPEP-UWM and machine learning. Training data were initially obtained using high-throughput techniques, and positive and negative datasets were generated. The predictive model was generated by calculating the explanatory variables of the peptides. To understand both site-specific and global characteristics, amino acid features (for site-specific characteristics) and peptide global features (for global characteristics) were generated. The constructed models were applied to the peptide database generated using BIOPEP-UWM, and bioactivity was predicted to explore candidate bile acid-binding peptides. Using this strategy, seven novel bile acid-binding peptides (VFWM, QRIFW, RVWVQ, LIRYTK, NGDEPL, PTFTRKL, and KISQRYQ) were identified. Our novel screening method can be easily applied to industrial applications using whole edible proteins. The proposed approach would be useful for identifying bile acid-binding peptides, as well as other BPs, as long as a large amount of training data can be obtained.

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“…Traditional approaches for protecting active peptides from enzymatic digestion capitalize on chemical modification of the peptide . These approaches include cyclization, lipidation, conjugation of PEG, introduction of unnatural amino acids, peptide backbone modification (e.g., N‐methylation), and capping of N‐ or C‐terminus, among others . Consequently, modified peptides are rendered inaccessible to, or unrecognizable by the active site of protease.…”

Section: Introductionmentioning

confidence: 99%

Proapoptotic Peptide Brush Polymer Nanoparticles via Photoinitiated Polymerization‐Induced Self‐Assembly

et al. 2020

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Herein, we report the photoinitiated polymerizationinduced self-assembly (photo-PISA) of spherical micelles consisting of proapoptotic peptide-polymer amphiphiles.T he one-pot synthetic approachy ielded micellar nanoparticles at high concentrations and at scale (150 mg mL À1)w ith tunable peptide loadings up to 48 wt. %. The size of the micellar nanoparticles was tuned by varying the lengths of hydrophobic and hydrophilic building blocks.C ritically,t he peptide-functionalized nanoparticles imbued the proapoptotic "KLA" peptides (amino acid sequence:KLAKLAKKLAKLAK) with two key properties otherwise not inherent to the sequence: 1) proteolytic resistance compared to the oligopeptide alone; 2) significantly enhanced cell uptake by multivalent displayof KLA peptide brushes.T he result was demonstrated improved apoptosis efficiency in HeLa cells.T hese results highlight the potential of photo-PISA in the large-scale synthesis of functional, proteolytically resistant peptide-polymer conjugates for intracellular delivery.

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Directed Evolution Using Stabilized Bacterial Peptide Display

Cited by 30 publications

References 74 publications

Chemical Diversification of Simple Synthetic Antibodies

Chemical Diversification of Simple Synthetic Antibodies

Machine learning screening of bile acid-binding peptides in a peptide database derived from food proteins

Proapoptotic Peptide Brush Polymer Nanoparticles via Photoinitiated Polymerization‐Induced Self‐Assembly

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