Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins.
In order to study 1) the mechanisms responsible for generating free ubiquitin monomer and 2) the function of ubiquitin carboxyl extension proteins in eukaryotes, we have developed a system for expression of human ubiquitin carboxyl extension proteins in prokaryotic and eukaryotic hosts. When expressed in Saccharomyces cerevisiae, the intact ubiquitin carboxyl extension proteins were rapidly processed to free ubiquitin monomer and extension protein. Furthermore, expression in this host conferred a slow growth phenotype mediated by the extension protein. Expression in Escherichia coli did not result in processing of the fusion proteins. However, when the expressed fusion proteins were purified from E. coli and incubated with a rabbit reticulocyte extract, the proteins were rapidly processed to free ubiquitin monomer and extension protein. These results show that human ubiquitin carboxyl extension proteins are processed to ubiquitin and extension protein when expressed in eukaryotic but not prokaryotic cells and that pre- and co-translational events are not necessary for their processing. Establishment of this system will allow for large scale purification of these proteins which will aid future studies on the function and structure of ubiquitin carboxyl extension proteins, as well as the mechanisms responsible for their processing.
Monia
B P
BP
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Ecker
D J
DJ
Jonnalagadda
S
S
Marsh
J
J
Gotlib
L
L
Butt
T R
TR
Crooke
S T
ST
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014452:Ubiquitins / Q000235:genetics* / Q000378:metabolism
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C045677:ubiquitin precursor