Protein Tags:How to Choose?@zoonbio

Summary

There are several affinity tags that can be used to simplify protein expression and purification. The choice of tag may depend on many different factors. The most common tag, the histidine tag, is often a (histidine)₆, but other polyhistidine tags consisting of between four and 10 histidine residues have been used. The latter provides for the strongest affinity for the chromatography medium. Other important tags are the GST and MBP tags, both of which are proteins, and Strep-tag II, which is a peptide optimized for chromatography on Strep-Tactin™ based chromatography media. Table on the following page highlights some key features of these tags.

What are the technical difficulties of e.coli system?

1.How to realize the expression of a large number of soluble proteins?
2.How to realize glycosylation modification of protein?

How much experience does zoonbio have for custom protein services in e.coli?

We started providing custom protein service since 2011. Till the end of 2019, more than 2,100 batches of custom proteins were successfully produced and delivered.

What starting materials do I have to provide ?

All we need to start with is the sequence information of your target protein/gene. We can start with gene synthesis with codon optimized for protein expression, and deliver you the final protein product. But if you would like us to start with your plasmid/virus, you are welcome to send them to us.

How much protein can your team usually obtain from 1L pilot project in e.coli expression system?

In bacteria system, the average is 1-5 mg/L. Our highest record is 200 mg/L.

What if the protein can't be expressed and purified in E. coli system?

There are a few things zoonbio scientists will do to solve the problem:
(1) Codon optimization is recommended if it has not been;
(2) remove the transmembrane region and signal peptide region;
(3) try to express truncated domains of the protein;
(4) we can also try different tags to increase the expression level;
(5) we may recommend customer to try different expression systems (e.g. insect cell system for certain proteins).

What tags does zoonbio recommend for protein expression and purification?

The common tags to use are GST, MBP. Trx, TF, MBP, Nus, SUMO, Flag, Fc, and so on. Zoonbio scientists may recommend different tags for different proteins based on the protein's property and the customer's specific requirements. His tag, either alone or fused with other tags (e.g. His-GST, etc.), is the most common one used for purification.

Tag-specific characteristics	Histidine tag	GST tag	MBP tag	Strep-tag II
Compatible expression systems	Can be used in any expression system.	Can be used in any expression system.	Can be used in any expression system.	Can be used in any expression system.
Metabolic burden to host	Low metabolic burden to expression host.	High metabolic burden to expression host.	High metabolic burden to expression host.	Low metabolic burden to expression host.
Yield after purification	Purification procedure gives high yields.	Purification procedure gives high yields.	Purification procedure gives high yields.	Purification procedure gives high yields.
Purity in a single step	Allows relatively high purity in a single purification step. Optimization of washing and elution conditions is recommended when extra high purity is needed in a single step.	Allows extremely high purity in a single purification step.	Allows extremely high purity in a single purification step.	Allows extremely high purity in a single purification step.
Effect on solubility of expressed protein	Does not enhance solubility.	May increase the solubility of the expressed protein.	May increase the solubility of the expressed protein.	Does not enhance solubility.
Purification products for different scales	Selection of purification products available for any scale.	Selection of purification products available for any scale.	Selection of purification products available for any scale.	Selection of purification products available for any scale.
Affinity tag removal	Small tag may not need to be removed (e.g., tag is weakly immunogenic so target protein can be used directly as an antigen for immunization). Site-specific proteases1enable cleavage of tag if required. TEV protease is often used to cleave off histidine tags. Note: Enterokinase sites that enable tag cleavage without leaving behind extra amino acids are preferable.	Site-specific protease (PreScission Protease)1 enables highly specific cleavage at 4ºC. This protease is also easily removed because it is itself GST-tagged (see Chapter 5).	Protease cleavage site can be engineered into the tagged protein.	Small tag may not need to be removed (e.g., tag is weakly immunogenic so the target protein can be used directly as an antigen in for immunization).
Tag detection	Histidine tag is easily detected using antiHis-based immunoassay.	GST tag is easily detected using a GST activity assay or anti-GST-based immunoassay.	Antibodies for detection available.	Antibodies for detection available.
Ease of purification	Simple purification. Note: Imidazole may cause precipitation in rare cases. Buffer exchange to remove imidazole may be necessary (see Chapter 11).	Simple purification. Very mild elution conditions minimize risk of damage to structure and function of the target protein. Buffer exchange may be desirable to remove reduced glutathione used for elution (see Chapter 11).	Simple purification. Very mild elution conditions minimize risk of damage to structure and function of the target protein. Buffer exchange may be desirable to remove maltose used for elution (see Chapter 11).	Simple purification. Very mild elution conditions minimize risk of damage to structure and function of the target protein. Buffer exchange may be desirable to remove desthiobiotin used for elution (see Chapter 11).
Elution conditions	Mild elution conditions.	Very mild elution conditions.	Very mild elution conditions.	Very mild elution conditions.
Suitability for dual tagging	Can be used for dual tagging to increase purity and to secure full-length polypeptides if the tags are placed at the N- and C-terminals. Dual tagging in combination with Strep-tag II minimizes effects on the target protein due to the small size of both tags.	Can be used for dual tagging to increase purity and to secure full-length polypeptides if the tags are placed at the N- and C-terminals.	Can be used for dual tagging to increase purity and to secure full-length polypeptides if the tags are placed at the N- and C-terminals.	Can be used for dual tagging to increase purity and to secure full-length polypeptides if the tags are placed at the N- and C-terminals. Dual tagging in combination with histidine tag minimizes effects on the target protein due to the small size of both tags.
Suitability for purification under denaturing conditions	Purification can be performed under denaturing conditions if required. Allows on-column refolding.	Cannot be used under denaturing conditions.	Cannot be used under denaturing conditions.	Cannot be used under denaturing conditions.
Effect on protease action	No effect on protease action.	A protein tag may hinder protease action on the target protein.	A protein tag may hinder protease action on the target protein.	No effect on protease action.
Effect on folding	Minimal effect on folding.	Believed to promote folding of recombinant proteins.	Believed to promote folding of recombinant proteins.	Minimal effect on folding.
Effect on structure and function of fusion partner	Small tag is less likely to interfere with structure and function of fusion partner.	Tagged proteins form dimers via the GST tag. A protein tag may interfere with structure and function of the target protein.	A protein tag may interfere with structure and function of the target protein.	Small tag is less likely to interfere with structure and function of fusion partner.
Effect on crystallization	Less risk of effects on crystallization than for large tags. May allow crystallization via coordination to Ni²⁺ ions.	May interfere with crystallization due to increased flexibility of the tagged protein. Removal of tag after purification may be needed. Crystals have been obtained in a few cases by using extra-short spacers between the tag and target protein.	May interfere with crystallization due to increased flexibility of the tagged protein. Removal of tag after purification may be needed. Crystals have been obtained in a few cases by using extra-short spacers between the tag and target protein.	Less risk of effects on crystallization than for large tags.
Suitability for purification of protein complexes	The tag will have a minimal effect on protein complex synthesis and will allow preparative purification of stable complexes provided that additional purification steps can be added for final purity. This tag is not suitable for tandem affinity chromatographic (TAP) analysis.	Suitable for protein complex purification requiring extremely mild wash and elution conditions.	Suitable for protein complex purification requiring extremely mild wash and elution conditions.	Suitable for protein complex purification requiring extremely mild wash and elution conditions.
Suitability for purification of proteins containing metal ions	Generally, not recommended for purification of proteins that contain metal ions.	Can be used for metal-containing proteins.	Can be used for metal-containing proteins.	Can be used for metal-containing proteins.