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Simplifying and streamlining Escherichia coli‐ based cell‐free protein synthesis
Author(s) -
Yang William C.,
Patel Kedar G.,
Wong H. Edward,
Swartz James R.
Publication year - 2012
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.1509
Subject(s) - cell free protein synthesis , t7 rna polymerase , escherichia coli , chloramphenicol acetyltransferase , reagent , chloramphenicol , protein biosynthesis , synthetic biology , lysis , chemistry , laboratory flask , biochemistry , biology , computational biology , bacteriophage , gene , antibiotics , gene expression , reporter gene
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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012