High yield expression of catalytically active USP18 (UBP43) using a Trigger Factor fusion system.
BACKGROUND
Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens.


RESULTS
High-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone.


CONCLUSIONS
The results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.
Department of Neuropathology, University of Freiburg, Freiburg, Germany.
Anja
A
Basters
Lars
L
Ketscher
Elke
E
Deuerling
Christoph
C
Arkona
Jörg
J
Rademann
Klaus-Peter
KP
Knobeloch
Günter
G
Fritz
D000818:Animals; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D043222:Ubiquitin Thiolesterase
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; C118306:Usp18 protein, mouse; D043222:Ubiquitin Thiolesterase; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase