Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 63, Issue 1, Pages 58-61Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.09.005
Keywords
Histidine tag of variable lengths; Ligation independent cloning; Immobilized metal ion affinity chromatography; T7 bacterial expression; High-throughput protein purification
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Funding
- National Institute of Health [GM 62412]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [P50GM062412] Funding Source: NIH RePORTER
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Immobilized metal ion affinity chromatography (IMAC) has become one of the most popular protein purification methods for recombinant proteins with a hexa-histidine tag (His-tag) placed at the C- or N-terminus of proteins. Nevertheless, there are always difficult proteins that show weak binding to the metal chelating resin and thus low purity. These difficulties are often overcome by increasing the His-tag to 8 or 10 histidines. Despite their success, there are only few expression vectors available to easily clone and test different His-tag lengths. Therefore, we have modified Escherichia coli T7 expression vector pET21 a to accommodate ligation-independent cloning (LIC) that will allow easy and efficient parallel cloning of target genes with different His-tag lengths using a single insert. Unlike most LIC vectors available commercially, our vectors will not translate unwanted extra sequences by engineering the N-terminal linker to anneal before the open reading frame, and the C-terminal linker to anneal as a His-tag. (c) 2008 Elsevier Inc. All rights reserved.
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