Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

Woodgate, John, Palfrey, David, Nagel, David, Hine, Anna V. and Slater, Nigel K.H. (2002). Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker. Biotechnology and Bioengineering, 79 (4), pp. 450-456.

Abstract

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5α fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the Smal site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins. © 2002 Wiley Periodicals, Inc.

Publication DOI: https://doi.org/10.1002/bit.10296
Divisions: Life & Health Sciences > Pharmacy
Life & Health Sciences > Biosciences
Life & Health Sciences
Uncontrolled Keywords: endotoxin,gene therapy,plasmid purification,protein-DNA interaction,zinc finger,Biotechnology,Microbiology
Full Text Link: http://www3.int ... 517689/abstract
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Date: 2002-08-20
Authors: Woodgate, John
Palfrey, David
Nagel, David
Hine, Anna V. ( 0000-0003-4065-831X)
Slater, Nigel K.H.

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