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

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About: A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : covidontheweb.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis
Creator	Shirouzu, Mikako Takemoto, Chie Hanada, Kazuharu Katsura, Kazushige Matsuda, Takayoshi Ohsawa, Noboru Sakamoto, Kensaku Tomabechi, Yuri Yonemochi, Mayumi
Source	PMC
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.
has issue date	2017-06-16(xsd:dateTime)
bibo:doi	10.1093/jb/mvx039
bibo:pmid	28992119
has license	no-cc
sha1sum (hex)	ae45992c96712b64942b02da4271ff90a6620671
schema:url	https://doi.org/10.1093/jb/mvx039
resource representing a document's title	A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis
has PubMed Central identifier	PMC7109869
has PubMed identifier	28992119
schema:publication	J Biochem
resource representing a document's body	covid:ae45992c96712b64942b02da4271ff90a6620671#body_text
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