Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering

Protein design is limited by the diversity of functional groups provided by the canonical protein „building blocks“. Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.     

In vivo incorporation of multiple noncanonical amino acids into proteins

Expansion of the standard genetic code enables the design of recombinant proteins with novel and unusual properties. Traditionally, such proteins have contained only a single type of noncanonical amino acid (NCAA) in their amino acid sequence. However, recently reported initial efforts demonstrate that it is possible with suppression-based methods to translate two chemically distinct NCAAs into a single recombinant protein by combining the suppression of different termination codons and nontriplet coding units (such as quadruplets). The possibility of expanding the code with various NCAAs simultaneously further increases the toolkit for the generation of multifunctionalized proteins.     

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.     

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl‐tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl‐tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA–aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

     

蛋白质功能化新策略:嵌入非天然氨基酸

天然蛋白质由20种天然氨基酸组成,这些蛋白质的构筑基元包含功能基团:羧基、氨基、巯基、硫醚、羟基、碱性胺、烷基和芳基。然而,这些有限的功能基团却不足以完成生物体内所有的生物学功能。为了更好地让生命的体现者--蛋白质完成更加精确和多样的生物学功能,自然界会对蛋白质进行翻译后的修饰,包括:磷酸化,甲基化,乙酰化或者羟基化,甚至在某些情况下,进化出一种新型的翻译机制以便插入硒代半胱氨酸或者吡咯霉素。受此启发,生物化学家发展出各种生物或化学方法来改变或插入新的蛋白质构筑基元,使天然蛋白质完成其相应的生物学功能或者使其具有某些特殊的性质,甚至是创造一种新酶。该文将简单介绍这些蛋白质修饰策略以及该领域的最新进展。     

Orchestrating the Biosynthesis of an Unnatural Pyrrolysine Amino Acid for Its Direct Incorporation into Proteins Inside Living Cells

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne‐bearing pyrrolysine amino acid was biosynthesized and incorporated site‐specifically allowing orthogonal double protein labeling.     

In Situ Deprotection and Incorporation of Unnatural Amino Acids during Cell‐Free Protein Synthesis

The S30 extract from E. coli BL21 Star (DE3) used for cell‐free protein synthesis removes a wide range of α‐amino acid protecting groups by cleaving α‐carboxyl hydrazides; methyl, benzyl, tert‐butyl, and adamantyl esters; tert‐butyl and adamantyl carboxamides; α‐amino form‐, acet‐, trifluoroacet‐, and benzamides; and side‐chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)‐4‐fluoroleucine, (S)‐4,5‐dehydroleucine, and (2S,3R)‐4‐chlorovaline into a protein through the direct use of their respective precursors, namely, (S)‐4‐fluoroleucine hydrazide, (S)‐4,5‐dehydroleucine hydrazide, and (2S,3R)‐4‐chlorovaline methyl ester. These results also show that the fluoro‐ and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.     

Proximity‐Enabled Protein Crosslinking through Genetically Encoding Haloalkane Unnatural Amino Acids

The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity‐enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity‐enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

A Facile Synthesis of N-Phosphoryl Amino Acids Containing Hydroxy Group

three kinds of N-(diisopropyloxyphosphoryl) amino acids containing hydroxyl group were prepared in high yield by using diisopropyl phosphite as the phosphorylating agent, sodium hypochlorite as the chlorinating agent and tetrabutyl ammonium bromide as the phase transfer catalyst in basic aqueous media.