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Scale-up, optimization, and analysis...

Stanford University.

Scale-up, optimization, and analysis of Escherichia coli extracts for cell-free protein synthesis.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Scale-up, optimization, and analysis of Escherichia coli extracts for cell-free protein synthesis.
作者:	Zawada, James Francis.
面頁冊數:	108 p.
附註:	Adviser: James R. Swartz.
附註:	Source: Dissertation Abstracts International, Volume: 65-09, Section: B, page: 4714.
Contained By:	Dissertation Abstracts International65-09B.
標題:	Engineering, Chemical.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3145586
ISBN:	0496045482

Scale-up, optimization, and analysis of Escherichia coli extracts for cell-free protein synthesis.
Zawada, James Francis.

Scale-up, optimization, and analysis of Escherichia coli extracts for cell-free protein synthesis. - 108 p.

Adviser: James R. Swartz.

Thesis (Ph.D.)--Stanford University, 2004.

As a result of this work, a new and useful fermentation procedure has been developed. In addition, the extract preparation protocol has been significantly improved, and the key steps have been identified. Together these advances both increase the feasibility of producing extract at a large scale and also contribute to the understanding of the process.

ISBN: 0496045482Subjects--Topical Terms:

226989
Engineering, Chemical.

Scale-up, optimization, and analysis of Escherichia coli extracts for cell-free protein synthesis.
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502 $a Thesis (Ph.D.)--Stanford University, 2004.
520 # $a As a result of this work, a new and useful fermentation procedure has been developed. In addition, the extract preparation protocol has been significantly improved, and the key steps have been identified. Together these advances both increase the feasibility of producing extract at a large scale and also contribute to the understanding of the process.
520 # $a Cell-free protein synthesis systems have been used for decades to study transcription and translation, and they continue to be used to produce proteins for biochemical experiments. However, the current procedures are limited to laboratory applications since they are not economically competitive with in vivo expression of proteins at a large scale. This is unfortunate because cell-free protein synthesis has advantages over in vivo systems which could accelerate the development of new pharmaceutical proteins. One central issue related to large scale in vitro protein production is the scale up of the extract preparation procedure. The current standard protocol is not much different from that used in 1963, and no systematic study of extract preparation has been performed.
520 # $a The extract preparation procedure was also investigated and improved. The centrifugation and runoff reaction steps were found to be the most important parts of the process. An improved procedure was developed which reduced material costs by 70% and shortened the duration of the process by almost 50%. Analysis of samples during extract preparation demonstrated that none of the steps result in a significant loss of ribosomes and that the association state of the ribosomal subunits does not impact the productivity of the extract.
520 # $a This work describes a novel fermentation procedure which sustains rapid growth (mu &sim; 0.7/hr) to moderate cell density (&sim;20 g/L DCW) without the accumulation of acetate. Extracts produced from these cultures were just as productive as extracts from standard low density batch cultures. Analysis of the extracts indicated that ribosome concentration and protein productivity both correlated with growth rate. In contrast, using amino acid enriched versus minimal medium did not affect protein productivity. In all extracts, only about 25% of the ribosomes were present as polysomes during cell-free protein synthesis and the total protein production corresponded to about 22 proteins per ribosome.
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