Browse
Submit Data
Databases
API
Help

Dataset Information

32 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids.

ABSTRACT: We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) N?-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N?-Boc-l-Lysine (BocK) and N?-propargyl-l-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.

SUBMITTER: Zhou H

PROVIDER: S-EPMC7644286 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids.

Zhou Han H Cheung Jenny W JW Carpenter Tomaya T Jones Stacey K SK Luong Nhu H NH Tran Nhi C NC Jacobs Savannah E SE Galbada Liyanage Sahan A SA Cropp T Ashton TA Yin Jun J

Bioorganic & medicinal chemistry letters 20191204 2

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) Nε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of Nε-Boc-l-Lysine (BocK) and Nε-propargyl-l-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrat ...[more]

PMID: 31836447

Similar Datasets

Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis.

Project description:New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNATyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.

| S-EPMC2825273 | biostudies-literature

Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells.

Project description: Not available

| S-EPMC11360239 | biostudies-literature

Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System.

Project description:Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.

| S-EPMC6481062 | biostudies-literature

Synthesis and Protein Incorporation of Azido-Modified Unnatural Amino Acids.

Project description:Two new azidophenylalanine residues (3 and 4) have been synthesized and, in combination with 4-azido-L-phenylalanine (1) and 4-azidomethyl-L-phenylalanine (2), form a series of unnatural amino acids (UAAs) containing the azide vibrational reporter at varying distances from the aromatic ring of phenylalanine. These UAAs were designed to probe protein hydration with high spatial resolution by utilizing the large extinction coefficient and environmental sensitivity of the azide asymmetric stretch vibration. The sensitivity of the azide reporters was investigated in solvents that mimic distinct local protein environments. Three of the four azido-modified phenylalanine residues were successfully genetically incorporated into a surface site in superfolder green fluorescent protein (sfGFP) utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity. SDS-PAGE and ESI-Q-TOF mass analysis verified the site-specific incorporation of these UAAs. The observed azide asymmetric stretch in the linear IR spectra of these UAAs incorporated into sfGFP indicated that the azide groups were hydrated in the protein.

| S-EPMC4603873 | biostudies-literature

Crucial optimization of translational components towards efficient incorporation of unnatural amino acids into proteins in mammalian cells.

Project description:The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA.

| S-EPMC3709914 | biostudies-literature

RF1 knockout allows ribosomal incorporation of unnatural amino acids at multiple sites.

Project description:Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but their low incorporation efficiency, which is possibly due to competition from release factors, limits the power and scope of this technology. Here we show that the reportedly essential release factor 1 (RF1) can be knocked out from Escherichia coli by 'fixing' release factor 2 (RF2). The resultant strain JX33 is stable and independent, and it allows UAG to be reassigned from a stop signal to an amino acid when a UAG-decoding tRNA-synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving new protein functions by enabling Uaa incorporation at multiple sites.

| S-EPMC3201715 | biostudies-literature

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.

Project description:Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more "orthogonal" to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.

| S-EPMC1482635 | biostudies-literature

Reagentless electrochemical biosensors through incorporation of unnatural amino acids on the protein structure.

Project description:Typical protein biosensors employ chemical or genetic labeling of the protein, thus introducing an extraneous molecule to the wild-type parent protein, often changing the overall structure and properties of the protein. While these labeling methods have proven successful in many cases, they also have a series of disadvantages associated with their preparation and function. An alternative route for labeling proteins is the incorporation of unnatural amino acid (UAA) analogues, capable of acting as a label, into the structure of a protein. Such an approach, while changing the local microenvironment, poses less of a burden on the overall structure of the protein. L-DOPA is an analog of phenylalanine and contains a catechol moiety that participates in a quasi-reversible, two-electron redox process, thus making it suitable as an electrochemical label/reporter. The periplasmic glucose/galactose binding protein (GBP) was chosen to demonstrate this detection principle. Upon glucose binding, GBP undergoes a significant conformational change that is manifested as a change in the electrochemistry of L-DOPA. The electroactive GBP was immobilized onto gold nanoparticle-modified, polymerized caffeic acid, screen-printed carbon electrodes (GBP-LDOPA/AuNP/PCA/SPCE) for the purpose of direct measurement of glucose levels and serves as a proof-of-concept of the use of electrochemically-active unnatural amino acids as the label. The resulting reagentless GBP biosensors exhibited a highly selective and sensitive binding affinity for glucose in the micromolar range, laying the foundation for a new biosensing methodology based on global incorporation of an electroactive amino acid into the protein's primary sequence for highly selective electrochemical detection of compounds of interest.

| S-EPMC9404255 | biostudies-literature

Toward efficient multiple-site incorporation of unnatural amino acids using cell-free translation system.

Project description:Amber suppression has been widely used to incorporate unnatural amino acids (UNAAs) with unique structures or functional side-chain groups into specific sites of the target protein, which expands the scope of protein-coding chemistry. However, this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein, which limits the development of unnatural proteins. To address this challenge, the suppression method using multiple termination codons (TAG, TAA or TGA) was proposed, and cell-free unnatural protein synthesis (CFUPS) system was employed. By the analysis of incorporating 3 different UNAAs (p-propargyloxy-l-phenylalanine, p-azyl-phenylalanine and L-4-Iodophenylalanine) and mass spectrometry, the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation. The CFUPS conditions were further optimized for better UNAA incorporation efficiency, including the orthogonal translation system (OTS) components, magnesium ions, and the redox environment. This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs, thereby synthesizing unnatural proteins with novel physicochemical functions.

| S-EPMC8718814 | biostudies-literature

Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids.

Project description:Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel-Crafts alkylation reactions and up to 83% ee for the product was achieved.

| S-EPMC5590542 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data