Cytiva™ Protein Select™ tag

Tag sequence and practical information

What is Cytiva Protein Select tag?

Cytiva
A tag that allows traceless self-cleavage Protein Protein of interest
Cytiva™ Protein Select™ tag is a 36-amino acid self-cleaving tag that enables Select tag
the chromatography affinity capture of recombinant proteins using Cytiva Unique affinity
Protein Select resin (coming soon). between the tag and
the ligand on the resin
During an affinity step performed with Cytiva Protein Select resin, the Ligand
protein self-cleaves from the tag and elutes with no residual tag amino acids.
When used in research, Cytiva Protein Select tag and resin simplify tagged
protein purification. During process development and later stages, they
standardize purification of any protein that does not have an affinity Cytiva Protein
binding partner. Select resin

Traceless self-cleavage of Cytiva Protein Select tag Fig 1. Cytiva Protein Select tag is recognized by the ligand
After expressing your protein containing Cytiva Protein Select tag, on Cytiva Protein Select resin.
collect and load your sample onto Cytiva Protein Select resin. Your target
protein binds to the resin ligand via the Cytiva Protein Select tag while
contaminants flow through during the wash. A folding event takes place
between the tag and the ligand, initiating autocleavage of the tag.
Your pure, tag-free protein self-cleaves from the tag and elutes with no residual
tag amino acids. You’ll have high purity in one simple chromatography step.

Sequence of Cytiva Protein Select tag

To benefit from Cytiva Protein Select technology, incorporate the Cytiva
Protein Select tag sequence and the gene of your protein of interest into
your expression plasmid (Fig 2). Insert the tag on the N-terminus of your
target protein (the autocleavage would not happen if the tag is inserted on
the C-terminus of the protein).

Here is the sequence of Cytiva Protein Select tag:

1 10 20 30
MVKIVSRKSLGVQNVYDIGVEKDHNFLLANGLIASN

cytiva.com
 Expression plasmid Protein Tagged protein
expression

Protein of
interest

Cytiva Protein Gene of the

Select tag protein of
sequence interest Cytiva Protein Select tag

Fig 2. Incorporate the Cytiva Protein Select tag sequence and the gene of your protein of interest into your
expression plasmid to obtain a protein tagged with Cytiva Protein Select tag.

Selection of the protein expression system

Cytiva Protein Select technology is flexible. You can use several different types
of expression systems (secreted and non-secreted) for production of your
recombinant protein.
Cytiva Protein Select tag has been expressed in mammalian (HEK and CHO) and
E.coli expression systems, and demonstrated no impact on expression levels of
target protein.

Automatic cleavage site

The cleavage site is between the last amino acid of the tag and the first amino acid of
the target protein (Fig 3). Automatic cleavage of the Cytiva Protein Select tag requires a
folding event with the ligand and thus limits the risk of premature traceless cleavage.

Cleavage
site

Protein of
Tag Ala–Ser–Asn aa1–aa2–aa3
interest

Last three amino First three amino

acids of Cytiva Protein acids of protein
Select tag of interest

Fig 3. The cleavage site of the Cytiva Protein Select tag is between the last three amino acids of the tag
and the first three amino acids of the target protein.
Practical considerations
Cleavage characteristics
The cleavage rate is mainly affected by the first 1–3 amino acids of the target protein,
in particular:
• Hydrophobic and aromatic amino acids in the second position will increase the
rate of cleavage.
• Small nonpolar, or negatively charged amino acids in the first position will slow
the rate of cleavage.
• Proline amino acids located in first and/or second position will halt the cleavage.
We suggest that you perform a point mutation of the proline amino acid(s).
Furthermore, cleavage may be affected by conditions such as temperature. It is
usually slower at lower temperatures.

If a signal peptide or a dual tag (e.g., 6 × His, FLAG) is desired upstream of the Cytiva
Protein Select tag, the initial Methionine (ATG codon) of the tag can be removed (Fig 4).

Cleavage site

Protein of
Signal peptide or additional tag Cytiva Protein Select tag
interest

Fig 4. An additional signal peptide or tag can be added if needed.

Hold step time

Once your sample is loaded onto a column packed with Cytiva Protein Select resin
and a wash step is done, a hold step (by pausing the flow) is performed during which
the cleavage takes place.
• A 4-hour hold step is recommended for cleavage, but an overnight hold step can
be performed.
• The procedure can be performed either at room temperatures or in a cold room.
• The hold time depends on the target protein.
 Need to learn
more on designing
recombinant
proteins?

Take our free More about Cytiva Protein

eLearning course Select technology at
cytiva.com/Protein-Select

Key features of Cytiva Protein Select resin and tag

• Simple affinity purification protocol for recombinant proteins.
• One-step purification and tag cleavage without the need for proteases
or other elution reagents.
• Delivers a highly pure, native protein with no remaining tag amino acids
after tag cleavage.
• Can be processed under mild conditions, which means no exposure to
conditions that cause protein aggregation or loss.
• Flexible, enabling the use in multiple expression systems.

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Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an
affiliate doing business as Cytiva. HiTrap and Protein Select are trademarks of Global Life
Sciences Solutions USA LLC or an affiliate doing business as Cytiva.
© 2023 Cytiva
For local office contact information, visit cytiva.com/contact

CY38322-12Oct23-FL