In:
BioTechniques, Future Science Ltd, Vol. 39, No. 3 ( 2005-09), p. 405-411
Abstract:
The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO 3 , yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15 N-, 13 C-labeled mineral salts and 2 H 2 O, the expressed polypeptides were highly ( 〉 90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.
Type of Medium:
Online Resource
ISSN:
0736-6205
,
1940-9818
Language:
English
Publisher:
Future Science Ltd
Publication Date:
2005
detail.hit.zdb_id:
1496354-1
SSG:
12
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