Photoactivatable Mussel‐Based Underwater Adhesive Proteins by an Expanded Genetic Code

Hauf, Matthias; Richter, Florian; Schneider, Tobias; Faidt, Thomas; Martins, Berta M.; Baumann, Tobias; Durkin, Patrick; Dobbek, Holger; Jacobs, Karin; Möglich, Andreas; Budiša, Nediljko

doi:10.1002/cbic.201700327

Cited by 80 publications

(93 citation statements)

References 48 publications

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“…As an alternative approach to enhance a production yield of Dopa‐MAPs, Hauf et al (2017) developed the highly active variant of MjTyrRS recognizing Dopa, allowing site‐specific incorporation of Dopa into multiple sites of protein. Here, in vivo residue‐specific incorporation of Dopa in tyrosine‐auxotrophic E. coli cells resulted in 4.19 mg/L of purified d fp‐3 with over 90% Dopa incorporation yield.…”

Section: Discussionmentioning

confidence: 99%

“…To expand protein space beyond the limit set by nature, several techniques to incorporate noncanonical amino acids into proteins have been developed (Jeon et al, 2018; Johnson, Lu, Van Deventer, & Tirrell, 2010; Liu & Schultz, 2010; Neumann, Wang, Davis, Garcia‐Alai, & Chin, 2010; Wang, Brock, Herberich, & Schultz, 2001; Won, Pagar, Patil, Dawson, & Yun, 2019). There have been recent advances in incorporating noncanonical amino acids into proteins; the design/selection of highly active orthogonal tRNA‐synthetases allowed for the efficient incorporation of a noncanonical amino acid into multiple sites of a target protein (Amiram et al, 2015; Hauf et al, 2017). Furthermore, the biosynthesis of noncanonical amino acids in situ has enabled sufficient intracellular supply, suitable for continuous translation (Voller & Budisa, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Enhanced production of Dopa‐incorporated mussel adhesive protein using engineered translational machineries

Jeong

Yang

et al. 2020

Biotech & Bioengineering

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Mussel adhesive proteins (MAPs) have great potential as bioglues, particularly in wet conditions. Although in vivo residue‐specific incorporation of 3,4‐dihydroxyphenylalanine (Dopa) in tyrosine‐auxotrophic Escherichia coli cells allows for production of Dopa‐incorporated bioengineered MAPs (dMAPs), the low production yield hinders the practical application of dMAPs. This low production yield of dMAPs is due to low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, to enhance the production yield of dMAPs, we investigated the coexpression of Dopa‐recognizing tyrosyl‐tRNA synthetases (TyrRSs). To use the Dopa‐specific Methanococcus jannaschii TyrRS (MjTyrRS‐Dopa), we altered the anticodon of tyrosyl‐tRNA amber suppressor into AUA (MjtRNATyrAUA) to recognize a tyrosine codon (AUA). Co‐overexpression of MjTyrRS‐Dopa and MjtRNATyrAUA increased the production yield of Dopa‐incorporated MAP foot protein type 3 (dfp‐3) by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of dfp‐3. Even with coexpression of Dopa‐recognizing TyrRSs, dfp‐3 has a high Dopa incorporation yield (over 90%) compared to ones prepared without TyrRS coexpression.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhanced production of Dopa‐incorporated mussel adhesive protein using engineered translational machineries

Jeong

Yang

et al. 2020

Biotech & Bioengineering

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“…Mm PylRS gene engineering was conducted via PCR using mutagenic primers with NNK (N = A, T, G, or C; K = G or T) randomizations at designated positions (S193, V401, and W417). Primers were synthesized by Merck, Darmstadt, Germany [ 28 , 60 ]. Primer sequences are listed in the Supplementary Materials (Note S2).…”

Section: Methodsmentioning

confidence: 99%

“…Primer sequences are listed in the Supplementary Materials (Note S2). The pBU16 plasmid containing pyrrolysyl–tRNA CUA driven by glnS’ promoter was digested with NdeI and KpnI [ 60 ]. The digested PCR products and linearized plasmid were ligated using T4 DNA ligase, and transformed into E. coli strain Top10.…”

Section: Methodsmentioning

confidence: 99%

“…To screen the Mm PylRS variants for activity towards Trp and Tyr analogs, the two plasmids, pBU16– MmPylRS–

and pET-28– sfGFP_R2TAG , were co-transformed into E. coli strain BL21(DE3) [ 60 ]. Single colonies were chosen from the plate and pre-cultured in 1 mL of LB medium supplemented with Amp (100 μg/mL) and Kan (100 μg/mL) at 37 °C overnight.…”

Section: Methodsmentioning

confidence: 99%

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Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

et al. 2020

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In protein engineering and synthetic biology, Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRS), with its cognate tRNAPyl, is one of the most popular tools for site-specific incorporation of non-canonical amino acids (ncAAs). Numerous orthogonal pairs based on engineered MmPylRS variants have been developed during the last decade, enabling a substantial genetic code expansion, mainly with aliphatic pyrrolysine analogs. However, comparatively less progress has been made to expand the substrate range of MmPylRS towards aromatic amino acid residues. Therefore, we set to further expand the substrate scope of orthogonal translation by a semi-rational approach; redesigning the MmPylRS efficiency. Based on the randomization of residues from the binding pocket and tRNA binding domain, we identify three positions (V401, W417 and S193) crucial for ncAA specificity and enzyme activity. Their systematic mutagenesis enabled us to generate MmPylRS variants dedicated to tryptophan (such as β-(1-Azulenyl)-l-alanine or 1-methyl-l-tryptophan) and tyrosine (mainly halogenated) analogs. Moreover, our strategy also significantly improves the orthogonal translation efficiency with the previously activated analog 3-benzothienyl-l-alanine. Our study revealed the engineering of both first shell and distant residues to modify substrate specificity as an important strategy to further expand our ability to discover and recruit new ncAAs for orthogonal translation

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Strategic Advances in Spatiotemporal Control of Bioinspired Phenolic Chemistries in Materials Science

Jia

Wen

Cheng

et al. 2021

Adv Funct Materials

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Nature-inspired phenolic chemistries have substantially nourished the engineering of advanced materials for widespread applications in energy, catalysis, and biomedicine, among others. To achieve predictable yet adaptable material structures and properties, the spatial and/or temporal control over the phenolic chemistries per se is increasingly demanded. In this review, a systematic overview of recent strategical advances in the spatiotemporal control of phenolic chemistries in materials science is given. The chemical diversity and reactivity of catechols and polyphenols are first introduced as phenolic building blocks. With a main focus on catechols (especially dopamine), renewed insights into their mechanisms of polymerization/assembly, adhesion, and cohesion are provided. The conditions for tuning phenolic polymerization/assembly are also outlined. This paper focuses on the latest strategies for imparting controlled manipulation of phenolic chemistries in terms of many aspects: growth kinetics, chemical compositions and structures, dimensions and architectures, spatial patterns, and surface functionality. Finally, critical issues facing this field with perspectives delivered on future research are discussed. As envisaged, this review will provide helpful guidance to orchestrate state-of-the-art phenolic chemistries and materials science for producing materials with intended, application-oriented properties and functions.

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Photoactivatable Mussel‐Based Underwater Adhesive Proteins by an Expanded Genetic Code

Cited by 80 publications

References 48 publications

Enhanced production of Dopa‐incorporated mussel adhesive protein using engineered translational machineries

Enhanced production of Dopa‐incorporated mussel adhesive protein using engineered translational machineries

Expanding the Scope of Orthogonal Translation with Pyrrolysyl-tRNA Synthetases Dedicated to Aromatic Amino Acids

Strategic Advances in Spatiotemporal Control of Bioinspired Phenolic Chemistries in Materials Science

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