Cotranslational incorporation of non‐standard amino acids using cell‐free protein synthesis

Quast, Robert B.; Mrusek, Devid; Hoffmeister, Christian; Sonnabend, Andrei; Kubick, Stefan

doi:10.1016/j.febslet.2015.04.041

Cited by 70 publications

(73 citation statements)

References 192 publications

(222 reference statements)

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“…Further, ncAA incorporation into proteins has opened the way to novel antibody drug conjugates (Axup et al, 2012; Zimmerman et al, 2014), modified human therapeutics (Cho et al, 2011), and protein biomaterials (Albayrak and Swartz, 2014), among other applications. With the ability to construct bio-based products beyond the limits of nature, expanding the genetic code has emerged as a major opportunity in synthetic and chemical biology (Des Soye et al, 2015; Hoesl and Budisa, 2012; Liu and Schultz, 2010; Quast et al, 2015). …”

Section: Introductionmentioning

confidence: 99%

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

Gan

Perez

Carlson

et al. 2017

Biotech & Bioengineering

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The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell’s natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology.

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Section: Introductionmentioning

confidence: 99%

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

Gan

Perez

Carlson

et al. 2017

Biotech & Bioengineering

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“…Cell-free protein synthesis (CFPS) is gaining ground as a method for ncAA incorporation, offering several advantages [7, 8]. First, CFPS can benefit from the relatively slower synthesis and the greater distance between ribosomes for proper protein folding [9].…”

Section: Introductionmentioning

confidence: 99%

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Gan

Fan

2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. Methods We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. Results By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Conclusions Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. General significance We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems.

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“…This eukaryotic cell-free system offers additional advantages in the form of native, ER-derived endogenous microsomes. Such microsomes contain the entire translocon machinery responsible for proper folding of MPs [8][9][10][11][12][13]. Recently, the potassium channel KcsA was synthesized successfully in this system [13].…”

Section: Introductionmentioning

confidence: 99%

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

Dondapati

Wüstenhagen

Kubick

2017

Methods in Molecular Biology

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Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cellfree synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

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Cotranslational incorporation of non‐standard amino acids using cell‐free protein synthesis

Cited by 70 publications

References 192 publications

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

Translation system engineering in Escherichia coli enhances non‐canonical amino acid incorporation into proteins

Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation

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