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Streamlining the preparation of "endotoxin-free" ClearColi cell extract with autoinduction media for cell-free protein synthesis of the therapeutic protein crisantaspase.
Hunt, J Porter; Zhao, Emily Long; Soltani, Mehran; Frei, Madison; Nelson, J Andrew D; Bundy, Bradley C.
Afiliação
  • Hunt JP; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
  • Zhao EL; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
  • Soltani M; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
  • Frei M; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
  • Nelson JAD; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
  • Bundy BC; Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
Synth Syst Biotechnol ; 4(4): 220-224, 2019 Dec.
Article em En | MEDLINE | ID: mdl-31890926
An "endotoxin-free" E. coli-based cell-free protein synthesis system has been reported to produce therapeutic proteins rapidly and on-demand. However, preparation of the most complex CFPS reagent - the cell extract - remains time-consuming and labor-intensive because of the relatively slow growth kinetics of the endotoxin-free ClearColiTMBL21(DE3) strain. Here we report a streamlined procedure for preparing E. coli cell extract from ClearColi™ using auto-induction media. In this work, the term auto-induction describes cell culture media which eliminates the need for manual induction of protein expression. Culturing Clearcoli™ cells in autoinduction media significantly reduces the hands-on time required during extract preparation, and the resulting "endotoxin-free" cell extract maintained the same cell-free protein synthesis capability as extract produced with traditional induction as demonstrated by the high-yield expression of crisantaspase, an FDA approved leukemia therapeutic. It is anticipated that this work will lower the barrier for researchers to enter the field and use this technology as the method to produce endotoxin-free E. coli-based extract for CFPS.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Guideline Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Guideline Idioma: En Ano de publicação: 2019 Tipo de documento: Article