Fusion protein/Tag

Fusion Tag /Protein

Definition:
A protein or a peptide located either on the C- or N- terminal of the target protein, which facilitates one or several of the following characteristics:

Improved solubility (S) - Fusion of the N-terminus of the target protein to the C-terminus of a soluble fusion partner often improves the solubility of the target protein.
Improved detection (D)- Fusion of the target protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) facilitates the detection of the resulting protein during expression or purification.
Improved purification (P)- Simple purification schemes have been developed for proteins used at either terminus which bind specifically to affinity resins.
Localization (L) - Tag, usually located on N-terminus of the target protein, which acts as address for sending protein to a specific cellular compartment.
Improved Expression (E)- Fusion of the N-terminus of the target protein to the C-terminus of a highly expressed fusion partner results in high level expression of the target protein.

Notice: The fusion protein must be continuous with the target protein - the same open reading frame must be maintained. Stop codons between the target protein and the fusion partner must be omitted.

Frequently Used Fusion Tags

Frequently Used Fusion Proteins

Additional Reading

Some Commercially Available Plasmids containing Various Fusion Tags/Proteins

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Frequently Used Fusion Tags

Name
(Link to sequence) Uses Location relative to target Protein
(N- or C-Terminal or Internal) Further Documentation Related Procedures
(See also Dr. Lebendiker's site)

His Tag
(6,8, or 10 amino acids)

Purification - Affinity Column: His tag binds very tightly [Kd~10^-13M) to immobilized divalent cations [e.g Ni⁺², Cu⁺², Zn⁺²]

Detection - Western Blot

N,C, I
NOVAGEN: pET manual (including methods of purifying many tags) (pdf)

NOVAGEN: Ni-NTA Hi-Bind Resins Protocols (pdf)

QUIAGEN: Ni-NTA purification manual (pdf)

QUIAGEN: NiNTA cell lysis under nature conditions

CLONTECH: Talon Resin Protocol (pdf)

CLONTECH: Talon Products (pdf)

AMERSHAM BIOSCIENCES Hi Tarp Chelating (pdf)

ACTIVE MOTIVE: Ni-Ted Profile (pdf)

AFFILAND: Metal Purification

Small scale His-Tag purification under nature conditions

Small scale His-Tag purification under denaturative conditions

Calbiochem - His Bind Purification Kits

T7 Tag
(11-16 amino acids)

Detection: Western Blot, Immunoprecipitation

Purification: Affinity Column (denaturing low pH elution needed)

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.

N, I NOVAGEN T7.Tag
Affinity Purification products (pdf)

S-Tag
(15 amino acids)

Detection: Western Blot,

quantitative Assay RNAse S assay possible for quantitative assay of expression levels.

Purification: Affinity Column

N, C, I S•Tag : A Multipurpose Fusion Peptide for Recombinant Proteins (pdf) S-Tag system (Novagen) (pdf)

Flag- Tag
(8 amino acids)

Purification

N, C Sigma's Flag Protein Expression System

HA Tag
(9 mino acids)

Detection

N

V5 Epitope
(15 amino acids)
I

Pel B

Localization: secretion signal

N

Xpress Epitope
(8 amino acids)

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Frequently Used Proteins

Name
(Link to sequence) Uses Location relative to target Protein
(N- or C-Terminal or Internal) Further Documentation Related Procedures
(See also Dr. Lebendiker's site)

GST
(Glutathione S Transferase)
(223amino acids, 26 kDa)

Solubility (lesser quality than Nus A or MBP) [N-term only]

Purification: glutathione affinity or GST antibody purification

Detection: Western Blot, Quantitative Assay (based on enzymatic activity)

N, C Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

CLONTECH GST products Handbook (pdf)

AMERSHAM BIOSCIENCES-GST fusion protein Handbook (pdf)

NOVAGEN GST.Bind Kits (pdf)

Small scale GST-fusion purification under nature conditions

MBP (Maltose Binding Protein)
(40kDa)

Solubility (better quality than GST or Trx) [N-term only]

Purification: Amylose affinity purification with maltose elution

N 1. E-coli MBP is uncommonly effective at promoting the solubilityof polypeptides to which it is fused
2. Smyth DR, Mrozkiewicz MK, McGrath WJ, Listwan P, Kobe B. Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 2003 Jul;12(7):1313-22. (reccommends addition of 3-6 ala-linker that stabilizes connection between target protein and MBP)
3. Routzahn KM, Waugh DS., Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins. J Struct Funct Genomics. 2002;2(2):83-92.
NEW ENGLAND BIOLAB: pMAL protein Fusion and Purification System

Nus A
(495 amino acids)

Solubility [N-term only]

N Expression of soluble heterologous proteins via fusion with NusA protein (pdf)

CBP (Calmodulin Binding Peptide)
(4kDa)

Purification

Detection

N, C
STRATAGENE: CBP Calmodulin-Binding Peptide Affinity Tag System (pdf)

GFP
(220 amino acids)

Detection: Immunofluoresence. Used as possible refoldind tag.

N, C Rapid protein-folding assay using GFP

Thioredoxin
(109amino acids, 11.7 kDa)

Solubility (lesser quality than Nus A or MBP) [N-term only]

Detection

Affinity purification with phenylarsine oxide-modified (ThioBond) resin

N, C
Using Rabbit Anti-Thioredoxin (fusion protein) Antibodies .
(Alpha Diagnostic International, Inc)

Mistic

Solubility (high level production of membrane proteins)

N Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S., NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science. 2005 ;307(5713):1317-21 (PDF)

Sumo

Solubility (high level production of membrane proteins)

N 1. Zuo X, Li S, Hall J, Mattern MR, Tran H, Shoo J, Tan R, Weiss SR, Butt TR. Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J Struct Funct Genomics. 2005;6(2-3):103-11 (PDF)
2. SUMO fusion technology for enhanced protein expression and purification in prokaryotes and eukaryotes.

DSBa

Solubility - better formation of S-S bonds.

N

1. Folding mechanisms of periplasmic proteins.
2. How proteins form disulfide bonds.

DSBc

Solubility - better formation of S-S bonds.

N Kurokawa Y, Yanagi H, Yura T. Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli. J Biol Chem. 2001 Apr 27;276(17):14393-9 (PDF)

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Additional Reading

Li Y., Self-cleaving fusion tags for recombinant protein production.Biotechnol. Lett. (2011); 33(5):869-81
Terpe K., Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 2003 ;60(5):523-33 (PDF)
Nilsson J, Stahl S, Lundeberg J, Uhlen M, Nygren PA., Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 1997 ;11(1):1-16 (PDF)
Bucher MH, Evdokimov AG, Waugh DS., Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallogr D Biol Crystallogr 2002 ;58(Pt 3):392-7 (PDF)
Smyth DR, Mrozkiewicz MK, McGrath WJ, Listwan P, Kobe B. Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 2003 Jul;12(7):1313-22. [Deals with MBP, GST and Trx fusions]
Fusion Tags Used for Recombinant Protein Expression and Purification, OR: Fusion tags used in recombinant protein expression and purification. Published in reference R.C. Stevens. "Design of high-throughput methods of protein production for structural biology " Structure, 8, R177-R185 (2000).

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