scholarly journals Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

10.3791/54273 ◽  
2016 ◽  
Author(s):  
Emanuel G. Worst ◽  
Matthias P. Exner ◽  
Alessandro De Simone ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Translation System ◽  
Noncanonical Amino Acids

Overproduction of noncanonical amino acids by Escherichia coli cells

Microbiology ◽  
2007 ◽  
Vol 76 (6) ◽  
pp. 712-718 ◽  
Author(s):  
E. V. Sycheva ◽  
T. A. Yampol’skaya ◽  
E. S. Preobrajenskaya ◽  
A. E. Novikova ◽  
N. G. Matrosov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Escherichia Coli Cells

Global substitution of hemeproteins with noncanonical amino acids in Escherichia coli with intact cofactor maturation machinery

2017 ◽  
Vol 106 ◽  
pp. 55-59
Author(s):  
Jan-Stefan Völler ◽  
Tuyet Mai Thi To ◽  
Hernan Biava ◽  
Beate Koksch ◽  
Nediljko Budisa
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Noncanonical Amino Acids

scholarly journals Directed evolution of rRNA improves translation kinetics and recombinant protein yield

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fan Liu ◽  
Siniša Bratulić ◽  
Alan Costello ◽  
Teemu P. Miettinen ◽  
Ahmed H. Badran
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Bacterial Population ◽  
Protein Yield ◽  
Rrna Sequence ◽  
Evolutionary Trajectory ◽  
Noncanonical Amino Acids ◽  
Rate Limiting

AbstractIn bacteria, ribosome kinetics are considered rate-limiting for protein synthesis and cell growth. Enhanced ribosome kinetics may augment bacterial growth and biomanufacturing through improvements to overall protein yield, but whether this can be achieved by ribosome-specific modifications remains unknown. Here, we evolve 16S ribosomal RNAs (rRNAs) from Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae towards enhanced protein synthesis rates. We find that rRNA sequence origin significantly impacted evolutionary trajectory and generated rRNA mutants with augmented protein synthesis rates in both natural and engineered contexts, including the incorporation of noncanonical amino acids. Moreover, discovered consensus mutations can be ported onto phylogenetically divergent rRNAs, imparting improved translational activities. Finally, we show that increased translation rates in vivo coincide with only moderately reduced translational fidelity, but do not enhance bacterial population growth. Together, these findings provide a versatile platform for development of unnatural ribosomal functions in vivo.

scholarly journals Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

2021 ◽  
Vol 12 ◽  
Author(s):  
Huawei Yi ◽  
Jing Zhang ◽  
Famin Ke ◽  
Xiurong Guo ◽  
Jian Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Expression ◽  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Noncanonical Amino Acids ◽  
E Coli ◽  
Growth Defects ◽  
Genetic Code Expansion ◽  
And Function

Incorporation of noncanonical amino acids (ncAAs) into proteins has been proven to be a powerful tool to manipulate protein structure and function, and to investigate many biological processes. Improving the yields of ncAA-containing proteins is of great significance in industrial-scale applications. Escherichia coli C321.ΔA was generated by the replacement of all known amber codons and the deletion of RF1 in the genome and has been proven to be an ideal host for ncAA-containing protein expression using genetic code expansion. In this study, we investigated the transcriptome and proteome profiles of this first codon reassignment strain and found that some functions and metabolic pathways were differentially expressed when compared with those of its parent strain. Genes involved in carbohydrate and energy metabolism were remarkably downregulated. Our results may provide important clues about the growth defects in E. coli C321.ΔA. Furthermore, we improved the yields of ncAA-containing proteins in E. coli C321.ΔA by integrating the T7 RNA polymerase system.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12

1993 ◽  
Vol 39 (12) ◽  
pp. 1155-1161 ◽  
Author(s):  
Alexandre Sasarman ◽  
Jaroslav Letowski ◽  
Guy Czaika ◽  
Volta Ramirez ◽  
Michael A. Nead ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Oxidase Activity ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Translation System ◽  
Protoporphyrinogen Oxidase ◽  
Escherichia Coli K12 ◽  
Amino Terminal

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21 202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription–translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.Key words: protoporphyrinogen oxidase (PROTOX), hemG gene, Escherichia coli, DPE herbicides, heme.

A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli

2010 ◽  
Vol 6 (4) ◽  
pp. 683 ◽  
Author(s):  
Ying Huang ◽  
William K. Russell ◽  
Wei Wan ◽  
Pei-Jing Pai ◽  
David H. Russell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Convenient Method ◽  
Noncanonical Amino Acids ◽  
Genetic Incorporation
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