Purification of recombinant proteins by chemical removal of the affinity tag.

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Ressource 1Download: serval:BIB_66D77565FEDE.P001 (661.87 [Ko])
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It was possible to publish this article open access thanks to a Swiss National Licence with the publisher.
Serval ID
serval:BIB_66D77565FEDE
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Purification of recombinant proteins by chemical removal of the affinity tag.
Journal
Applied Biochemistry and Biotechnology
Author(s)
Rais-Beghdadi C., Roggero M.A., Fasel N., Reymond C.D.
ISSN
0273-2289 (Print)
ISSN-L
0273-2289
Publication state
Published
Issued date
1998
Volume
74
Number
2
Pages
95-103
Language
english
Abstract
The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.
Keywords
Amino Acid Sequence, Animals, Chromatography, Affinity, Cyanogen Bromide/chemistry, Escherichia coli, Histidine/chemistry, Mass Spectrometry, Molecular Sequence Data, Plasmodium falciparum, Protozoan Proteins/biosynthesis, Protozoan Proteins/chemistry, Recombinant Proteins/biosynthesis, Recombinant Proteins/isolation &amp, purification
Pubmed
Web of science
Open Access
Yes
Create date
24/01/2008 15:02
Last modification date
01/10/2019 6:18
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