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An efficient system for high‐level expression and easy purification of authentic recombinant proteins
Author(s) -
Catanzariti AnnMaree,
Soboleva Tatiana A.,
Jans David A.,
Board Philip G.,
Baker Rohan T.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04618904
Subject(s) - fusion protein , ubiquitin , escherichia coli , cleave , recombinant dna , biochemistry , biology , target protein , enzyme , histidine , tandem affinity purification , protein expression , computational biology , chemistry , microbiology and biotechnology , affinity chromatography , gene
Abstract Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine‐tagged ubiquitin fusions, and a histidine‐tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high‐throughput applications.