Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.
Biotechnol Prog
; 28(2): 413-20, 2012.
Article
em En
| MEDLINE
| ID: mdl-22275217
ABSTRACT
Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biossíntese de Proteínas
/
Proteínas Recombinantes
/
Fracionamento Celular
/
Proteínas de Escherichia coli
Tipo de estudo:
Evaluation_studies
Idioma:
En
Revista:
Biotechnol Prog
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos