Metal-Responsive Regulation of Enzyme Catalysis using Genetically Encoded Chemical Switches

Zubi, Yasmine S.; Seki, Kosuke; Liu, Ying; Hunt, Andrew J.; Liu, Bingqing; Roux, Benoît; Jewett, Michael C.; Lewis, Jared C.

doi:10.26434/chemrxiv-2021-9ql74

Cited by 2 publications

(3 citation statements)

References 57 publications

(72 reference statements)

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“…pET.BCS-BpyRS was cloned as previously described using Gibson Assembly (38). Expression plasmids for the Chimeric pylRS, the M. alvus pylRS, and the Lum1 pylRS were synthesized in the pET.BCS backbone by Twist Biosciences.…”

Section: Methodsmentioning

confidence: 99%

“…Lysate was then aliquoted into single-use aliquots, flash frozen in liquid nitrogen, and stored at -80 °C. aaRS Purification pET.BCS-BpyRS was cloned as previously described using Gibson Assembly (38). Expression plasmids for the Chimeric pylRS, the M. alvus pylRS, and the Lum1 pylRS were synthesized in the pET.BCS backbone by Twist Biosciences.…”

Section: Dna Purificationmentioning

confidence: 99%

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Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

Seki

Galindo

Jewett

2022

Preprint

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A wide variety of non-canonical amino acids (ncAAs) can be incorporated into proteins through the coordinated action of a stop codon suppressing tRNA and aminoacyl-tRNA synthetase. However, methods to discover and characterize suppressor tRNAs are generally lacking. In this work, we show that cell-free systems can express functional suppressor tRNAs using endogenous machinery and characterize their activity. This method is compatible with widely used orthogonal tRNAs, such as the Methanocaldococus jannaschii tyrosyl tRNA, the Methanosarcina barkeri pyrrolysyl tRNA, the Methanomethylophilus Alvus pyrrolysyl tRNA, and an engineered Int pyrroysyl tRNA. Modifying the workflow to evaluate TAA suppression revealed that the M. jannaschii and M. alvus are highly functional TAA suppressors in cell-free systems. Finally, we show that we can express two distinct tRNAs simultaneously, enabling the incorporation of multiple, distinct ncAAs. In total, our work shows that cell-free systems are useful platforms to express and characterize tRNAs.

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Section: Methodsmentioning

confidence: 99%

Section: Dna Purificationmentioning

confidence: 99%

Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

Seki

Galindo

Jewett

2022

Preprint

Self Cite

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“…The genetic encoding of noncanonical amino acids (ncAAs) in proteins enables molecular-level manipulation of native protein structure and function as well as the introduction of new functions, properties, or chemical entities (Chin, 2014; Chung et al, 2020; Islam et al, 2021; Lewis et al, 2021; Shandell et al, 2021; Vanderschuren et al, 2022; Wang et al, 2009; Zubi et al, 2022). The number of ncAAs that can be encoded during protein biosynthesis and the number of organisms in which they can be encoded continues to expand (Dumas et al, 2015; Vargas-Rodriguez et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Lahiri

Deventer

2022

Preprint

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Proteins containing noncanonical amino acids (ncAAs) provide opportunities for dissecting fundamental biological processes and engineering protein therapeutics with diverse chemistries. Incorporation of more than one ncAA into a single construct can endow a protein with multiple useful features, such as unique molecular recognition and covalent crosslinking. Herein, for the first time, we encode two chemically distinct ncAAs into proteins prepared in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems (OTSs): E. coli tyrosyl-tRNA synthetase/tRNATyr, E. coli leucyl-tRNA synthetase/tRNALeu, and M. alvus pyrrolysyl-tRNA synthetase/tRNAPyl. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Further, we found that multiple factors impacted TGA readthrough efficiency in a manner analogous to TAG readthrough, including the local nucleotide context surrounding stop codons and deletion of selected genes from the yeast genome. In contrast, the identity of the suppressor tRNA affected TGA readthrough efficiency quite differently than TAG readthrough efficiency. Altogether, these observations facilitated the systematic investigation of dual ncAA incorporation in response to TAG and TGA codons within both intracellular and yeast-displayed protein constructs. Employing a proficient combination of OTSs, pairs of ncAAs could be encoded within both protein formats with efficiencies up to 6% of wildtype protein controls. Moreover, exploration of dual ncAA incorporation in yeast display format aided in demonstrating important applications of doubly-substituted proteins. First, dual ncAA incorporation within a simple synthetic antibody construct resulted in retention of antigen binding functionality, indicating that the doubly-substituted proteins can retain their basic functions. Second, presentation of reactive alkyne and azide functionalities within yeast-displayed constructs enabled sequential installation of two distinct chemical probes using bioorthogonal reactions--strain-promoted- and copper-catalyzed azide-alkyne cycloadditions (SPAAC and CuAAC)--on the yeast surface. This confirmed the ability to chemoselectively modify yeast-displayed proteins at two distinct sites. Furthermore, by utilizing a soluble form of a doubly substituted construct, we validated the feasibility to selectively double label proteins in solution in a single pot reaction. Overall, our work facilitates the addition of a 22nd amino acid to the yeast genetic code and provides an important toolkit that expands the scope of applications of ncAAs for basic biological research and drug discovery in yeast.

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Metal-Responsive Regulation of Enzyme Catalysis using Genetically Encoded Chemical Switches

Cited by 2 publications

References 57 publications

Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

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