Hi,
I would like to point out, for the sake of
fairness, that thrombin (high quality bovine thrombin such as sold by HTI for
example) is still much cheaper *to use*
than commercial TEV. One milligram of TEV, TVMV, AcTEV, and so forth can be
used to cleave anywhere in between 10 to 100 mg of ‘reasonably sized*’
fusion protein, with typical ratio of around 1:25**. Thrombin is considerably
more processive – 1 microgram of thrombin typically cleaves about 1 mg of
fusion protein (again, reasonably sized) which is a ratio of 1:1000. The
differences in processivity can be explained in terms of enzyme biochemistry
and structure. These days everyone uses David Waugh’s stabilizing
mutation(s) of the TEV protease which in its native form deactivates itself in
a few hours by means of site-specific autoproteolysis. TVMV protease does not
undergo this process.
Of course, TEV and other potyviral
proteases offer unique advantages such as resistance to most protease
inhibitors (yes, even E-64), extreme fidelity, and so forth. But the cheapest
way to get them is to make them in-house (it’s a 2-step procedure
yileding about 100-150 mg of TEV or TVMV protease per liter of E. coli).
Artem
* all other things equal, in molar terms,
the efficiency is the same for large or small constructs; but in weight terms larger
fusions may be cleaved with higher efficiency. On the other hand, in practical
terms large fusions can be tough to cleave due to the cleavage site steric protection
by the sheer bulk of the construct.
** GST-TEV or MBP-TEV proteases can be
even less efficient due to steric hindrance
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of Chun Luo
Sent: Friday, February 08, 2008
12:31 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Tag, you're
in! Flag, V5, etc
Yongfu,
Small peptide tags usually don’t
interfere with protein folding and linker is not required. With removable tags,
the protease site also serves as a linker. In most cases, putting a protease cleavage
site such as TEV or PreScission site is good enough. The proteases (HRV3C and
TurboTEV) are now available at a cost less than using thrombin (http://www.accelagen.com/proteins.htm#protease).
So you can remove the tag and the proteases easily.
For detection with Western blot, Flag, HA,
Myc-tags are good and His-tag may be problematic since not all anti-His
antibodies work well. However, His-tag gives an option to do binding in
denatured condition. So for purification purpose, His-tag is probably your best
bet.
If you know that the precise N- or
C-terminus is critical for protein function, put the tag on the other end or
use Factor Xa cleavage site to remove N-terminal tag. You’ll be surprised
to find out that many proteins can be easily purified without using any tag.
When we did the purification of SARS 3C protease, the purification scheme
without using tag was actually simpler with higher yield. We have purified
proteins for crystallography without using affinity tag from insect cells at an
expression level of ~1 mg/L. From E. coli, a 3-step purification is generally
sufficient to purify protein without using affinity tag.
Chun
Chun Luo, Ph.D.
The
Protein Expert
Accelagen,
Inc.
TeL:
858-350-8085
ext
111
Fax:
858-350-8001
[log in to unmask]
www.accelagen.com
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of Y. -F. Li
Sent: Thursday, February 07, 2008
1:29 PM
To: [log in to unmask]
Subject: [ccp4bb] Tag, you're in!
Flag, V5, etc
Hello,
I'd appreciate it if anyone could provide information (experiences or
publications) on the following:
1. Put a tag such as FLAG, V5, etc etc, at the N- and/or C-termini, in
order for specific detection, but not interfering with protein
folding/structure;
2. Is a linker between the tag and target protein needed? What linkers
(length, specific sequences) would you suggest?
3. What are the choices of such tags and why would you recommend them?
Thank you for your time and sharing in advance.
Yongfu Li