Dear Colleagues,
I agree that the Strep-tag II provides a high end tool compared to
the His-tag. In our hands the resin - i.e. StrepTactin affinity
matrix either home-made or commercially provided by IBA
http://www.iba-go.de/prottools/prot_fr01_01.html - shows exceptional
performance:
- It has high specific protein binding capacity, escpecially if one
bears in mind that much of the capacity of a metal chelate column is
wasted by host cell proteins that usually compete with the His-tagged
protein.
- It shows excellent reproducibility, also because no addition of
imidazole as well as high salt to the protein extract or preelution
steps of non-specifically bound host cell proteins have to be
optimized.
- The matrix is extremely robust, at least if not charged with
polluted extracts or high amounts of biotin. For example, we have
been using a preparative StrepTactin column (50 mL bed volume) for
more than two years with several hundred purification runs.
In addition, one should keep in mind that, contrasting with the
His-tag, this method does not interfere with metal ions (either bound
to the protein of interest or when used for crystallization screens).
Also, the Strep-tag chromatography does not affect free Cys residues,
which are prone to oxidation catalyzed by Cu(II) or Ni(II).
Thus, we consider the Strep-tag II to be extremely helpful in
particular for protein crystallization and, in fact, we have
published crystal structures for a series of proteins using this
tool. In conclusion, I always recommend to use the Strep-tag II first
when purifying a new recombinant proteins because success is almost
guaranteed (provided that soluble protein is present at all).
Regarding use of the Strep-tag technique, I may draw your attention
to a recent review article in Nature Protocols:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17571060&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
I will be happy to provide PDF reprints.
Best regards,
Arne Skerra
>3) STREP tag vs His tag - kind of like comparing a sports car to a lorry.
>Sometimes you win using one but not the other - however for a generic case I
>would nearly always prefer the truck. STREP can be essentially one-step, but
>the yields are often quite poor due to low resin capacity and fragility; the
>resin is expensive and does not always regenerate well. In comparison,
>His-tag nearly always works, usually gives loads of protein (as much as your
>system can express in a competent form), gives adequate purity (if you use
>His-Select or Talon in combination with detergent lysis you get only a
>couple of foreign bands) and is cheap to use and easy to regenerate. So, if
>you're working with something crazy or esoteric - some 250 kDa protein that
>only expresses in the cerebrospinal fluid of recombinant Madagaskar Chipmunk
>embryos - then STREP tag is probably better. For an 'easy' protein expressed
>in bacteria, baculovirus, or yeast - His tag is a reasonable default
>starting point.
>
>Artem
>
>-----Original Message-----
>From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
>Anirban Adhikari
>Sent: Thursday, December 20, 2007 5:56 PM
>To: [log in to unmask]
>Subject: [ccp4bb] Question about protein expression (not CCP4 related)
>
>Hello,
>
>Sorry about the non CCP4 related question. I have few questions regarding
>protein expression:
>
...
>3. Is anyone purifying their protein using the Strep-tag II/Strepactin
>affinity system and if so how does it compare to His-tag?
>
>I am looking forward to reading your opinions and suggestions.
>
>Thanks a lot,
>Anirban
>
>Anirban Adhikari, Ph. D.
>Postdoctoral Research Scholar
>Department of Molecular Biology
>UT Southwestern Medical Center
>5323 Harry Hines Blvd,
>Dallas, TX 75390-9063
--
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Prof. Dr. Arne Skerra [log in to unmask]
Lehrstuhl f. Biologische Chemie Phone: +49 (0)8161 71-4351
Technische Universitaet Muenchen Fax: -4352
85350 Freising-Weihenstephan
Germany http://www.wzw.tum.de/bc
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