An efficient system for high-level expression and easy purification of authentic recombinant proteins.
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.
Catanzariti
Ann-Maree
AM
Molecular Genetics Group, Division of Molecular Medicine, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia.
Soboleva
Tatiana A
TA
Jans
David A
DA
Board
Philip G
PG
Baker
Rohan T
RT
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D028884:Karyopherins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics*
D028884:Karyopherins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D005982:Glutathione Transferase