Published in reference R.C. Stevens. "Design of high-throughput methods of protein production for structural biology " Structure, 8, R177-R185 (2000).

Tag

Size

Fusion tag
location

Tag type

Comments

His-tag

6, 8, or 10 aa

N-, C-, internal

purification

Most common purification tag used for immobilized metal affinity chromatography (IMAC) one-step purification [81]. Purification possible even under denaturing conditions [82]. Tag possibly influences crystallization

T7-tag

11 or 16 aa

N-, internal

purification, enhanced expression

Monoclonal antibody-based purification (denaturing low pH elution needed). Leaves unnatural N-terminal amino acids on the recombinant protein. Possibly enhanced expression levels since the T7-tag is derived from the T7 gene 10 which is the naturally most abundant phage T7 gene product.

S-tag

15 aa

N-, C-, internal

Purification and detection

S-protein (104 aa, Ribonuclease A minus S-tag peptide sequence) modified resin affinity purification. RNAse S assay possible for quantitative assay of expression levels.

FLAG™ peptide

(DYKDDDDK)

8 aa

N-, C-

Purification

Ca2+-dependent monoclonal antibody affinity purification with EDTA elution. Tag cleavable with enterokinase [83].

thioredoxin

109 aa

(11.7 kDa)

N-,C-
 

Purification and enhanced expression
 

Affinity purification with phenylarsine oxide-modified (ThioBond) resin.

His-patch thioredoxin

109 aa

(11.7 kDa)

N-,C-

Purification and enhanced expression
 

Use of His-patch modified thioredoxin for IMAC affinity purification [84].

lacZ (β-Galactosidase)

116 kDa

N-,C-

Purification

Purification using p-amino-phenyl-b-D-thiogalactoside-modified sepharose. Classical tag used for protecting peptides from proteolytic degradation. However, fusion proteins with this tag have a high tendency to be insoluble. Active enzyme is a tetramer

chloramphenicol acetyltransferase

24 kDa

N-

Secretion, purification and detection

Chloramphenicol-sepharose purification. Enzymatic assay possible for quantitation.

trpE

27 kDa

N-

Purification

Often form insoluble precipitates. Hydrophobic interaction chromatographic purification.

avidin/streptavidin/
Strep-tag

 

 

Purification and secretion

Biotin affinity purification and streptavidin affinity purification (Strep-tag) [85].

T7gene10

260 aa

N-

Purification and enhanced expression

Produces insoluble fusion protein (potential enhanced expression for toxic clones).
 

staphylococcal protein A
 

14 kDa (or 31 kDa)

N-

Purification and Secretion
 

IgG antibody affinity purification possible (denaturing low pH elution needed). Fusion protein secretion due to protein A signal sequence [86].

streptococcal protein G

28 kDa

N-, C-

Purification and Secretion

Albumin affinity purification, low pH elution needed. Fusion protein secretion due to protein G signal sequence.

glutathione-S-transferase (GST)
 

26 kDa

N-

Purification

Glutathione affinity or GST antibody purification. Enzymatic activity assay possible for quantitative analysis. Fusion proteins form dimers.

dihydrofolate reductase (DHFR)
 

25 kDa
 

N-

Purification

Methotrexate-linked agarose used for purification.

cellulose binding domains (CBD's)
 

156 aa/
114 aa/
107 aa

N-/
N-/
C-
 

Purification and secretion

Cellulose-based resins used for affinity purification with water elution [87,88]. Different constructs available for cytoplasmic or periplasmic expression. Fusion proteins susceptible to proteolysis between the fusion partners [89].

maltose binding protein (MBP)
 

40 kDa

N-, C-

Purification and secretion

Amylose affinity purification with maltose elution.

galactose-binding protein
 

 

 

Purification

Galactose-sepharose purification.

calmodulin binding protein (CBP)
 

4 kDa

N-, C-

Purification and detection

Calmodulin/Ca2+-affinity purification with EDTA elution. Can potentially assay expression levels with 32P-cAMP kinase.

hemagglutinin influenza virus (HAI)

 

 

Purification

 

green fluorescent protein (GFP)

220 aa

N-, C-

Detection

Used as reporter gene fusion for detection purposes [90]. Used at one time for possible refolding tag.

HSB-tag

11 aa

C-

Purification

Monoclonal antibody-based purification (denaturing low pH elution needed).

B-tag (VP7 protein region of bluetongue virus)

 

 

Purification

Anti-BTag antibody purification.

polyarginine

5-15 aa

C-

Purification

S-sepharose (cationic resin) purification. Fusion proteins potentially insoluble.

polycysteine

4 aa

N-

Purification

Thiopropyl-sepharose purification.

polyphenyalanine
 

11 aa

N-

Purification

Phenyl-superose (hydrophobic interaction chromatography) purification.

(Ala-Trp-Trp-Pro)n

 

 

Purification

 

polyaspartic acid
 

5-16 aa
 

C-

Purification

Anionic resin purification.

KSI

125 aa

N-

Enhanced expression

High-level inclusion body production.

c-myc

 

 

Purification

Anti-myc antibody purification.

ompT
/ompA
/pelB
/DsbA
/DsbC

22 aa /21 aa /20 aa /208 aa (21.8 kDa) /236 aa
 

N-

Secretion

Periplasmic leader sequences for potential protein export and folding [91], as well as potential disulfide bond formation and isomerization.

chitin binding domain

 

N-, C-

Expression

Used in the Impact™ system, with intein-based expression constructs

NusA

495 aa (54.8 kDa)

N-

Possible enhanced solubility

Potentially improve solubility for proteins that are overexpressed.

ubiquitin

76 aa

N-

Possible enhanced solubility

lac operator affinity purification

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