Incorporation of unnatural amino acids using a washed cell extract in a cell-free protein synthesis system

민승의,김동명 한국공업화학회 2014 한국공업화학회 연구논문 초록집 Vol.2014 No.1

The ability to incorporate unnatural amino acids can expand the scope of protein engineering beyond the present limitations of using 20 canonical amino acids. While cell-based production of unnatural amino acid-containing protein is cumbersome and time and labor-intensive process, herein we propose a rapid and efficient tool for translational incorporation of unnatural amino acids into proteins. The advantages of cell-free protein synthesis include speed and throughput in protein production. In addition, the open nature of cell-free protein synthesis allows direct modifications of the components for protein synthesis. While the simple replacements of amino acids led to mixed synthesis of proteins containing unnatural and natural amino acids, in our experiments to replace tyrosine with 3,4-dihydroxy-L-phenylalanine (L-DOPA), almost exclusive incorporation of unnatural amino acids was achieved by washing the cell extract to remove residual natural amino acids in cell extract. Proposed strategy should be able to provide a facile route to selective incorporation unnatural amino acids in protein structure.

An efficient method to incorporate unnatural amino acids in recombinant proteins by reassigning a sense codon

이병성,유태현 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1

The protein synthesis system has been engineered to incorporate unnatural amino acid into proteins, and this has opened up new routes for engineering proteins with novel compositions. While such systems have been applied in research, the current methods have limitations, and there remains a need to develop new approaches with respect to the wider application of unnatural amino acids. In this study, we reported a strategy for incorporating unnatural amino acids into proteins by reassigning one of the Arg sense codons, the AGG codon. Using this method, several unnatural amino acids were quantitatively incorporated into the AGG site. Furthermore, we applied the method to multiple AGG sites, and even to tandem AGG sequences. The method developed and described here could be used for engineering proteins with diverse unnatural amino acids, particularly when employed in combination with other methods.

Importance of Expression System in the Production of Unnatural Recombinant Proteins in Escherichia coli

Niraikulam Ayyadurai,Rameshkumar Neelamegam,Soundrarajan Nagasundarapandian,Selvakumar Edwardraja,Hyung Soon Park,Soo Jae Lee,Tae Hyeon Yoo,Hyungdon Yoon,이선구 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.3

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system

Asymmetric Sythesis of Unnatural L-Amino Acids Using Thermophilic Aromatic L-Amino Acid Transaminase

Cho, Byung-Kwan,Seo, Joo-Hyun,Kim, Ju-Han,Lee, Chang-Soo,Kim, Byung-Gee The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.4

Aromatic L-amino acid transaminase is an enzyme that is able to transfer the amino group from L-glutamate to unnatural aromatic ${\alpha}-keto$ acids to generate ${\alpha}-ketoglutarate$ and unnatural aromatic L-amino acids, respectively. Enrichment culture was used to isolate thermophilic Bacillus sp. T30 expressing this enzyme for use in the synthesis of unnatural L-amino acids. The asymmetric syntheses of L-homophenylalanine and L-phenylglycine resulted in conversion yields of >95% and >93% from 150 mM 2-oxo-4-phenylbutyrate and phenylglyoxylate, respectively, using L-glutamate as an amino donor at $60^{\circ}C$. Synthesized L-homophenylalanine and L-phenylglycine were optically pure (>99% enantiomeric excess) and continuously pre-cipitated in the reaction solution due to their low solubility at the given reaction pH. While the solubility of the ${\alpha}-keto$ acid substrates is dependent on temperature, the solubility of the unnatural L-amino acid products is dependent on the reaction pH. As the solubility difference between substrate and product at the given reaction pH is therefore larger at higher temperature, the thermophilic transaminase was successfully used to shift the reaction equilibrium toward rapid product formation.

Creation of (R)-beta transaminase not yet found in nature by directed evolution of D-amino acid aminotransferase

Hyungdon YUN 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Optically pure unnatural amino acids including beta- and gamma- amino acids are crucial active intermediates frequently used in the industrial synthesis of a range of chemicals and pharmaceuticals. Over time, numerous biocatalytic routes catalyzed by lipase, amidases, acylases, nitrilases, hydantoinases, ammonia lyases, aldolases, and amino acid dehydrogenase have been developed to access to chiral beta- and g- amino acids. Each enzyme catalyzed reaction has its advantages and limitations. The development of an efficient biocatalytic process for chiral unnatural amino acids remains a challenging task. The toolbox of transaminase (TA) is rapidly expanding, since they possess many benefits over other enzymes including broad substrate specificity, high enantioselectivity and no requirement for cofactor regeneration. beta-TA can reversibly transfer amino group from beta- and gamma-amino acids as well as amines onto amine acceptor such as pyruvate. To the best of our knowledge naturally occurring (R)-beta-TAs reactive for various beta - and gamma-amino acids has not yet been discovered, despite many (S)-beta-TAs have been reported. Therefore, creating (R)-beta-TA with broad substrate specificity and high catalytic efficiency is desperately needed for the synthesis of chiral beta- and gamma- amino acids. we have successfully created an (R)- beta-TA, not yet found in nature through the hybridizing of DATA and (R)-ATA created from DATA, and subsequent ISM-based directed evolution of the hybrid.

Production of Chiral Amines and Unnatural Amino Acids Using ω-Transaminases

윤형돈 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.1

The development of broadly applicable catalytic methods for the sustainable production of chiral amines and unnatural amino acids has been identified as a key research priority by the pharmaceutical industry. Among different biocatalysts, ω-transaminases (ω-TAs) have recently received a great deal of attention as promising catalysts due to their ability to produce a wide range of optically pure amines and unnatural amino acids. ω-TA reaction has many desirable features when compared to other enzymes such as hydrolases and dehydrogenases, including broad substrate specificity, high enantioselectivity, high turnover number, and no requirement for regeneration of external cofactors. Here, our recent efforts to produce optically pure unnatural amino acid by using ω-TAs will be presented.

Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis

Mathew, S.,Bea, H.,Nadarajan, S.P.,Chung, T.,Yun, H. Elsevier Science Publishers 2015 Journal of biotechnology Vol.196 No.-

<P>Optically pure beta-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of beta-amino acids continues to pose a major challenge for researchers. omega-Transaminase has emerged as an important class of enzymes for generating amine compounds. However, only a few omega-transaminases have been reported so far which show activity towards aromatic beta-amino acids. In this study, (S)-omega-transaminase from Burkholderia graminis C4D1M has been functionally characterized and used for the production of chiral aromatic beta-amino acids via kinetic resolution. The enzyme showed a specific activity of 3.1 U/mg towards rac-beta-phenylalanine at 37 degrees C. The K-m and K-cat values of this enzyme towards rac-beta-phenylalanine with pyruvate as the amino acceptor were 2.88 mM and 91.57 min(-1) respectively. Using this enzyme, racemic beta-amino acids were kinetically resolved to produce (R)-beta-amino acids with an excellent enantiomeric excess (> 99%) and similar to 50% conversion. Additionally, kinetic resolution of aromatic beta-amino acids was performed using benzaldehyde as a cheapamino acceptor. (C)2015 Elsevier B.V. All rights reserved.</P>

Enhanced production of unnatural amino acid-containing proteins in a cell-free protein synthesis system

이경호,김동명,Christy Catherine 한국공업화학회 2016 Journal of Industrial and Engineering Chemistry Vol.37 No.-

Replacement of canonical amino acids with unnatural amino acids (UAAs) can provide proteins withnovel physicochemical properties and biological functions. In this study, as an alternative option toconventional cell-based methods, we used a cell-free protein synthesis system as a flexible platform forfacile and efficient production of UAA-containing proteins. We designed a cell-free protein synthesissystem derived from the extract of Escherichia coli cells to maximize the selective incorporation of UAAsinto the protein structure. First, for the purpose of avoiding competitive incorporation of canonicalamino acids and UAAs, the cell extract was extensively washed using a diafiltration process to removeresidual amino acids, thereby making the protein synthesis reaction completely dependent upon theexogenous addition of amino acids. In addition, the relatively low affinity of UAAs for cognate aminoacyl-tRNA synthetase was kinetically overcome by increasing the concentration of UAAs to nonphysiologicallevels. As a result of these modifications of the cell-free synthesis systems, we were able to produce UAAcontainingproteins at comparable yields to those of proteins made of canonical amino acids.

Enhanced production of unnatural amino acid-containing proteins in a cell-free protein synthesis system

Lee, K.H.,Catherine, C.,Kim, D.M. Korean Society of Industrial and Engineering Chemi 2016 Journal of industrial and engineering chemistry Vol.37 No.-

<P>Replacement of canonical amino acids with unnatural amino acids (UAAs) can provide proteins with novel physicochemical properties and biological functions. In this study, as an alternative option to conventional cell-based methods, we used a cell-free protein synthesis system as a flexible platform for facile and efficient production of UAA-containing proteins. We designed a cell-free protein synthesis system derived from the extract of Escherichia coli cells to maximize the selective incorporation of UAAs into the protein structure. First, for the purpose of avoiding competitive incorporation of canonical amino acids and UAAs, the cell extract was extensively washed using a diafiltration process to remove residual amino acids, thereby making the protein synthesis reaction completely dependent upon the exogenous addition of amino acids. In addition, the relatively low affinity of UAAs for cognate aminoacyl-tRNA synthetase was kinetically overcome by increasing the concentration of UAAs to nonphysiological levels. As a result of these modifications of the cell-free synthesis systems, we were able to produce UAA-containing proteins at comparable yields to those of proteins made of canonical amino acids. (C) 2016 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.</P>

Cloning-independent engineering of elastin-like polypeptides using unnatural amino acids

( Christy ),오수진,민승의,원종인,윤현동,김동명 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1

Based on the central dogma of protein synthesis, traditional methods for protein engineering require that altering protein structure and function must be accompanied by changing the nucleotide sequence of the genes encoding the protein. However, the preparation of a template gene for each individual protein requires a great deal of time and effort, thereby limiting the throughput and scope of studying engineered proteins. In this study, we describe translation-level engineering of proteins using cell-free protein synthesis. Taking advantage of the promiscuity of aminoacyl tRNA synthetases in accepting structurally similar amino acid analogues, unnatural amino acids were introduced into elastin-like polypeptides in place of the corresponding cognate amino acids. Through the incorporation of various analogues and starting from the same gene, the phase transition temperatures of elastin-like polypeptides became tunable. Our results demonstrate the usefulness of cell-free protein synthesis for protein engineering using unnatural amino acids without the need for cloning.