High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

ABSTRACTSynthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacteriumEscherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system inE. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

Applied and Environmental Microbiology ◽

10.1128/aem.00433-14 ◽

2014 ◽

Vol 80 (12) ◽

pp. 3576-3584 ◽

Cited By ~ 10

Author(s):

Gaoyan Wang ◽

David C. Manns ◽

John J. Churey ◽

Randy W. Worobo

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Bacillus Thuringiensis ◽

Gene Cluster ◽

Expression Vector ◽

Expression System ◽

Structural Genes ◽

Cry Protein ◽

Content Type ◽

Thurincin H

ABSTRACTThurincin H is an antimicrobial peptide produced byBacillus thuringiensisSF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1,thnA2, andthnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designatedB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter,thnA1, and a Cry protein terminator into theEscherichia coli-B. thuringiensisshuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in thethnA1gene, were generated and separately transformed intoB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities ofB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3carrying different pGW133 variants against three different indicator strains were subsequently compared.

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Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

Chemical Communications ◽

10.1039/d1cc04611j ◽

2021 ◽

Author(s):

Babu Sudhamalla ◽

Anirban Roy ◽

Soumen Barman ◽

Jyotirmayee Padhan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Protein Interactions ◽

Site Specific ◽

Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

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Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

Author(s):

Sviatlana Smolskaya ◽

Yaroslav Andreev

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Nonsense Suppression ◽

Expression Vectors ◽

Site Specific ◽

Suppressor Trna ◽

E Coli ◽

Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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Improving orthogonal tRNA-synthetase recognition for efficient unnatural amino acid incorporation and application in mammalian cells

Molecular BioSystems ◽

10.1039/b904228h ◽

2009 ◽

Vol 5 (9) ◽

pp. 931 ◽

Cited By ~ 46

Author(s):

Jeffrey K. Takimoto ◽

Katrina L. Adams ◽

Zheng Xiang ◽

Lei Wang

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1

Journal of the American Chemical Society ◽

10.1021/ja5069728 ◽

2014 ◽

Vol 136 (44) ◽

pp. 15577-15583 ◽

Cited By ~ 123

Author(s):

Wolfgang H. Schmied ◽

Simon J. Elsässer ◽

Chayasith Uttamapinant ◽

Jason W. Chin

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation

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Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells

Chemistry & Biology ◽

10.1016/s1074-5521(03)00124-8 ◽

2003 ◽

Vol 10 (6) ◽

pp. 573-580 ◽

Cited By ~ 40

Author(s):

Sarah L Monahan ◽

Henry A Lester ◽

Dennis A Dougherty

Keyword(s):

Amino Acids ◽

Unnatural Amino Acids ◽

Mammalian Cells ◽

Site Specific

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Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

Journal of Biological Chemistry ◽

10.1074/jbc.m707355200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1525-1533 ◽

Cited By ~ 113

Author(s):

Shixin Ye ◽

Caroline Köhrer ◽

Thomas Huber ◽

Manija Kazmi ◽

Pallavi Sachdev ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

G Protein ◽

Unnatural Amino Acids ◽

Unnatural Amino Acid ◽

G Protein Coupled Receptors ◽

Calcium Flux ◽

Unnatural Amino Acid Mutagenesis ◽

Amino Acid Mutagenesis ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

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