Hi Gloria,
I asked for a little baker's yeast from a lab in our department and PCRed
both the SMT3 domain and the SUMOase gene from its genomic DNA, then cloned
them to E coli vectors. The good thing about yeast is that most of its genes
are single exon so one do not really need to find cDNA for most of its
proteins.
My SUMOase construct was based on the structure pdb 1EUV. It included a
N-term His6 followed by a bamHI site and the SUMOase 421-601. The sumo
protease construct can be produced from BL21 at maybe 10-20mg/L and behaved
quite well. It worked for me when I did cleavage test. However I didn't use
it extensively due to some problem specific to my protein domain.
SMT3
ETHINLKVSDGSSEIFFKIK
KTTPLRRLMEAFAKRQGKEM
DSLRFLYDGIRIQADQTPED
LDMEDNDIIEAHREQIG
His6-SUMO protease
MHHHHHHGSKKLVPELNEKD
DDQVQKALASRENTQLMNRD
NIEITVRDFKTLAPRRWLND
TIIEFFMKYIEKSTPNTVAF
NSFFYTNLSERGYQGVRRWM
KRKKTQIDKLDKIFTPINLN
QSHWALGIIDLKKKTIGYVD
SLSNGPNAMSFAILTDLQKY
VMEESKHTIGEDFDLIHLDC
PQQPNGYDCGIYVCMNTLYG
SADAPLDFDYKDAIRMRRFI
AHLILTDALK
Zhijie
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From: "Gloria Borgstahl" <gborgst...@gmail.com>
Sent: Thursday, May 24, 2012 3:41 PM
To: <CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] SUMO(ULP-1) protease
My fellow crystallographers,
We are thinking the SUMO/His vectors would be nice to have in the lab
aresenal... but. The stumbling block is that the protease needed for
cleavage is very expensive at crystallography scale. SUMO(ULP-1)
protease costs ~$700/mg fusion protein. It would not be a problem
for labs using micrograms of protein, but is prohibitive at our level
of protein purification.
Has anyone found a way around this? Your pal, Gloria
