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Fundamentals of Modern Peptide Synthesis
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
Vauquelin and Robique (1806) isolated the first amino acid from Asparagus sativus; they named the amino acid asparagine (Vauquelin and Robique 1806). After glycine was discovered from gelatin hydrolysate (Braconnot 1820) Rose et al. described threonine from food ingredients in 1935 (Rose et al. 1935). Later, the last two of the 22 proteinogenic amino acids, selenocysteine and pyrrolysine, were discovered in 1986 (Zinoni et al. 1986) and 2002 (Srinivasan et al. 2002).
Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences
Published in mAbs, 2020
Gargi Roy, Jason Reier, Andrew Garcia, Tom Martin, Megan Rice, Jihong Wang, Meagan Prophet, Ronald Christie, William Dall’Acqua, Sanjeev Ahuja, Michael A Bowen, Marcello Marelli
The incorporation of a nnAA requires engineered cells expressing an orthogonal aaRS/tRNA pair with specificity for the nnAA. In an effort to generate a highly efficient system, we generated stable platform hosts expressing the pyrrolysine tRNA synthetase (pylRS) and its cognate tRNA (tRNApyl) derived from archaebacterial Methanosarcina mazei, an enzyme previously shown to mediate nnAA incorporation in mammalian cells.18,54 Stable cell lines armed with this system are a desirable tool for manufacturing because it represents the starting point for the construction of expression cell lines. First, we constructed a vector bearing expression cassettes for PylRS under the control of a CMV promoter, as well as 18 tandem repeats of tRNA under the U6 snRNA promoter. The vector also contained a puromycin selection marker for stable cell line selection. Following transfection into AstraZeneca-proprietary suspension CHO cells, the transfectants were plated in 96-well plates and subjected to selection in puromycin-containing media.73,74 Surviving colonies were expanded and screened for amber suppression activity using a high throughput functional assay. This required the transfection of an mRNA reporter probe encoding a red fluorescent protein (RFP)-green fluorescent protein (GFP) fusion containing an amber codon between the two genes (RFPamb-GFP) (Figure 1a).75,76 Transfected cells were exposed to the nnAA (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Figure 2a, AzK) to enable amber suppression. With this probe, all transfected cells express RFP, but only cells capable of amber suppression (and nnAA incorporation) also express the GFP reporter (Figure 1b).
Challenges and promise at the interface of metaproteomics and genomics: an overview of recent progress in metaproteogenomic data analysis
Published in Expert Review of Proteomics, 2019
Henning Schiebenhoefer, Tim Van Den Bossche, Stephan Fuchs, Bernhard Y. Renard, Thilo Muth, Lennart Martens
One potential issue is the occurrence of untypical amino acids such as selenocysteine and pyrrolysine by certain microbial species. As the codons encoding these amino acids are often stop codons in many organisms that do not incorporate these amino acids, the in-silico generation of amino acid sequences from nucleotide-sequencing data will result in prematurely terminated sequences. The downstream analysis may also be influenced by these amino acids when not provided to the software tool of choice (e.g. search engines, annotation software). For example, the consequence might be that sequences containing these rare amino acids cannot be identified.
An overview of lantibiotic biosynthetic machinery promiscuity and its impact on antimicrobial discovery
Published in Expert Opinion on Drug Discovery, 2020
The incorporation of non-canonical amino acids (ncAA) has been applied to increase the diversity of lantibiotics. In this method the orthogonal translation system (OTS) is developed to aminoacylate a tRNA with ncAA, which is then added site specifically to a growing peptide chain. This has successfully been applied to the lantibiotic nisin using stop codon suppression (SCS), a method that allows reprogramming of the amber stop codon TAG, replacing it with a sense codon to add the ncAA. The stop codon TAG was selected as it is the least frequently used stop codon in the nisin producing strain Lactococcus lactis and less likely to have a significant fitness cost to the host cell. To accomplish this the heterologous expression of nisin in E. coli via the construction of a T7 promotor based set up for the recombinant expression of nisABC was applied. To enable translation of the ncAA the pyrrolysine (Pyl) tRNA sythetase and corresponding tRNA from the archaeon Methanosarcina mazei [pyrrolysyl-tRNA sythetase (PylRS)-tRNAPyl] was employed. This tRNA synthetase recognizes its cognate tRNA and naturally charges it with the amino acid Pyl, allowing the incorporation into the growing peptide chain. The Pyl system has previously been used in other non-bacterial systems including yeasts [61] and mammalian cells [62] for the incorporation of ncAA. For the incorporation of ncAA into nisin a system using E.coli containing the biosynthetic machinery for recombinant production of nisin that incorporated the ncAA Nε-Boc-L-Lysine (BocK). The genetic code of the nisin producer strain Lactococcus lactis was expanded by the introduction of PylRS-tRNAPyl pair to allow SCS. BocK was then incorporated into nisin at different locations and the effect on the antimicrobial activity assessed. Screening of nisin amber codon libraries identified the most efficient sites for incorporation of ncAA that elicits high antimicrobial activity. Results identified two promising candidates nisin (14BocK) and nisin (K12BocK), both of which displayed high antimicrobial activity against the indicator strain, whereas little or no inhibition was observed without the incorporation of ncAA [63].