Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Smith, Mark T.; Hawes, Anna Katz; Shrestha, Pragya; Rainsdon, Jay Marvin; Wu, Jeffrey C.; Bundy, Bradley C.

doi:10.1016/j.procbio.2013.10.012

Cited by 27 publications

(23 citation statements)

References 47 publications

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“…In situ expression of o‐tRNA using ribozyme‐based method (Albayrak and Swartz, ) was recently reported and achieved increased yields. Since, it has also been shown that it is possible to achieve incorporation of UAAs using the Methanocaldococcus jannaschii ( Mj ) Mj ‐tyrosyl‐aminoacyl‐tRNA synthetase/tRNA orthogonal pair (Smith et al, ).…”

Section: Introductionmentioning

confidence: 99%

Genetically expanded cell‐free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Chemla

Ozer

Schlesinger

et al. 2015

Biotech & Bioengineering

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Cell-free protein synthesis offers a facile and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from a DNA template. At the same time, genetic code expansion methods are gaining attention due to their ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins via ribosomal translation. These systems are based on the exogenous addition of an orthogonal translation system (OTS), comprising an orthogonal tRNA, and orthogonal aminoacyl tRNA synthetase (aaRS), to the cell-free reaction mixture. However, these components are unstable and their preparation is labor-intensive, hence introducing a major challenge to the system. Here, we report on an approach that significantly reduces the complexity, effort and time needed to express UAA-containing proteins while increasing stability and realizing maximal suppression efficiency. We demonstrate an endogenously introduced orthogonal pair that enables the use of the valuable yet insoluble pyrrolysyl-tRNA synthetase in a cell-free system, thereby expanding the genetic repertoire that can be utilized in vitro and enabling new possibilities for bioengineering. With the high stability and efficiency of our system, we offer an improved and accessible platform for UAA incorporation into proteins.

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Section: Introductionmentioning

confidence: 99%

Genetically expanded cell‐free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Chemla

Ozer

Schlesinger

et al. 2015

Biotech & Bioengineering

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“…Three different media formulations and varying levels of NaCl were tested: LB‐Miller (the recommended media for ClearColi protein preparations), 2xYT media (reported to produce high‐yielding CFPS extracts), and TB media (which supports E. coli growth to higher cell densities) . The three media formulations were evaluated at increasing NaCl levels, as enhanced ClearColi growth has been suggested at higher osmolarity and ionic strength .…”

Section: Resultsmentioning

confidence: 99%

“…Cell extracts were made using either E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA) or ClearColi BL21 DE3 (Lucigen, Middleton, WI). Extract was produced as described previously with either LB, 2xYT, or TB fermentation media …”

Section: Methodsmentioning

confidence: 99%

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

et al. 2018

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Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

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“…[160] Recent applications of ncAA incorporation in E. colibased cell-frees ystems include the development of novel therapeutics,p olymers, and enzymes( reviewed in refs. [37]and [161]). In particular,t he cell-free synthesis of homogenous antibody-drug conjugates (an antigen-binding IgG coupled to ac hemotherapeutic cytotoxin) is ap romising tool to generate highly specific cancer therapeutics.…”

Section: Extractsfrom Culturedh Uman Cell Linesmentioning

confidence: 99%

ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

et al. 2016

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Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Cited by 27 publications

References 47 publications

Genetically expanded cell‐free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Genetically expanded cell‐free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

ChemInform Abstract: Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

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