A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

Abstract

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.

CDK1 dependent phosphorylation of hTERT contributes to cancer progression

“…In vitro kinase assay using recombinant hTERT proteins. For recombinant protein expression using cell-free synthesis-coupled transcription-translation 66 , the target cDNA fragment, corresponding to 191-306 residues of hTERT was subcloned into the pCR2.1-TOPO vector (Thermo Fisher Scientific). Proteins, subjected to affinity purification, were N-terminally fused with a modified natural Fig.…”

The NF‐κB regulator IκBβ exhibits different molecular interactivity and phosphorylation status from IκBα in an IKK2‐catalysed reaction

“…For recombinant protein expression using Escherichia coli , and for cell‐free synthesis‐coupled transcription–translation , the target cDNA fragments, IκBα full length, corresponding to residues 1–317 of human IκBα, and IκBβ full length, corresponding to residues 1–356 of human IκBβ, were subcloned into the pCR2.1‐TOPO vector (Thermo Fisher Scientific), which is modified to allow expression of target proteins fused with a modified natural polyhistidine N11‐tag (amino acid sequence: MKDHLIHNHHKHEHAHAEH) with a tobacco etch virus (TEV) protease recognition site and a GSSGSSG linker sequence .…”

“…T7 promoter‐driven expression and cell‐free protein synthesis of N‐terminal affinity‐tagged, N11‐IκBα and N11‐IκBβ were performed in an E. coli S30 extract in S30 buffer (10 m m Tris/acetate buffer at pH 8.2, containing 60 m m potassium acetate, 16 m m magnesium acetate and 1 m m DTT) .…”

Reduced Efficacy of a Src Kinase Inhibitor in Crowded Protein Solution

“…The gene encoding human Src kinase domain (residues 260-533) was cloned into the pCR2.1 vector (Invitrogen) and expressed as a fusion with N-terminal histidine and GST tags using Escherichia coli cellfree reaction supplemented with chaperones and YopH 70,71 . The protein was purified by affinity chromatography using a HisTrap column (GE Healthcare) and further by ion exchange on a HiTrap Q column (GE Healthcare) and used for in vitro kinase assay.…”