3.

Maltose Binding Protein (MBP)

The maltose-binding protein (MBP) was one of the affinity tags to be used for
the purposes of overcoming problems associated with the expression and
purification of fusion proteins. Generally, recombinant proteins tagged with MBP
can alleviate toxicity and improve expression level and protein solubility (Zhao, Li,
& Liang, 2013). MBP tagging may produce a higher percentage of recombinant
protein than that the polyhistidine tag does. However, the disadvantage of MBP is
the size and immunogenicity of the affinity tag, which complicates any downstream
application.
The purification of MBP-tagged proteins is achieved by conventional amylose
resin-based chromatography (Zhao et al., 2013). The elution of the MBP-fused
proteins is at neutral pH using mild maltose-containing buffer conditions (Zhao et
al., 2013). MBP tag is effective when placed on the N-terminal or C-terminal end
of target proteins. MBP tag fused to either the N or C terminur has been shown to
increase recombinant soluble expression, a common approach is to fuse it to the N-
terminus. Unfortunately, since no tag can be ideal for all applications, to increase
the versatility of tags and downstream applications, combinatorial-tagging methods
have been developed for various needs (Needle & Waugh, 2014). Two affinity tags
expressed in tandem along with a protease cleavage site preceding the target
protein, enables the use of multiple purification strategies (Needle & Waugh,
2014). By incorporating a solubility-enhancing tag in combination with a
purification tag, improvements in protein solubility and expression yields as well
as methods for efficient purification are achieved (Austin, Nallamsetty, & Waugh,
2009) .

Figure 3. 1 A general schematic for construct design strategy-affinity/ solubility tag combination
at the N-terminus
Source: (Austin et al., 2009)
 This is the example about protein purification of E.coli using MBP tag. The
pMal Protein Fusion and Purification System allows researches to use E.coli to
create a fusion between an xpressed target protein and MBP, and purify the fusion
protein in a single step. A gene of interest is cloned into a pMAL vector downstream
of the malE gene that encodes MBP. The strong Ptac promoter and MBP translation
initiation signals control expression of the gene. Upon induction, this system fuses
the target protein sequence with a portion of MBP to create a fusion protein that is
isolated using amylose affinity chromatography. Different pMAL vectors permit
fusion proteins to be secreted into the periplasm or produced in the cytoplasm, and
each introduce a protease cleavage site between MBP and the target protein to
facilitate tag removal.

Figure 3. 2 Step of using MBP as tag for protein purification

Source: (BioLabs, 2016)
MBP fusion proteins can also be expressed in yeast. The pKLMF-series vectors
permit intracellular expression of MBP fusions in the yeast Kluyvermyces lactis.
These vectors stably integrate into the host chromosome and utilize the strong yeast
LAC4 promoter for expression. Both vectors also introduce a protease cleavage site
between MBP and the target protein (BioLabs, 2016).
4. Glutathione S-Transferase (GST)
The glutathione 𝑆-Transferase (GST) tag is another well established affinity tag
based on the strong affinity of GST for immobilized glutathione. The GST tag is
the best suitable for use in prokaryotic expression, because GST is a family
cytosolic proteins that are present in eukaryotic organism but generally not found
in bacteria (Zhao et al., 2013). GST is a 211 amino acid protein (26 kDa) whose
expressed in the pGEX plasmid vector. The result of expression from this vector is
a GST-tagged fusion protein in which the functional GST protein (26 kDa) is fused
to the N-terminus of the recombinant protein (Simons, 1977). Depending on the
vector, many have a thrombin or other protease recognition site. Depending on the
protein concentration (typically high levels) GST can dimerize. Using 1-chloro-
2,4-dinitrobenze (CNDB) as a substrate, GST activity can be followed using a
simple colormetric assay with glutathione.
Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for
glutathione S-transferase (GST). When reduced glutathione is immobilized through
its sulfhydryl group to a solid support, such as cross-linked beaded agarose, it can
be used to capture pure GST or GST-tagged proteins via the enzyme-substrate
binding reaction.

Figure 3. 3 Immobilized Glutathione. Cross linked beaded agarose bound to reduce glutathione
Source:(ThermoFIsher, 2014)
 Binding is most effective in near-neutral buffers (physiologic conditions) such
as Tris-buffered saline (TBS) pH 7.5. Because binding depends on preserving the
essential structure and enzymatic function of GST, protein denaturants are not
compatible.
After washing an affinity column to remove non-bound sample components,
the purified GST-fusion protein can be dissociated and recovered (eluted) from a
glutathione column by addition of excess reduced glutathione. The free glutathione
competitively displaces the immobilized glutathione binding interaction with GST,
allowing the fusion protein to emerge from the affinity column. This affinity system
commonly yields greater than 90% pure GST-tagged recombinant protein from
crude bacterial or mammalian cell lysate samples. Glutathione-based affinity
purification of GST-tagged fusion proteins is easily done at either small, medium
or large scales to produce microgram, milligram or gram quantities.
Similar to the MBP tag, GST tag has long been used to increase the solubility
of fusion proteins in E. coli. GST tagged proteins are captured by immobilized
glutathione and then are eluted under mild, non-denaturing conditions using
reduced glutathione. The general purification strategy is thus to bind the GST
fusion protein on a column of immobilized glutathione, wash away all the other
stuff, and then elute the protein. There are two steps to GST fusion protein
purification. First, the GST fusion protein is separated from all other proteins by
running the supernatant over a glutathione column; the GST fusion protein binds
to the glutathione column and all other proteins are washed away. The GST protein
is the eluted from the column with glutathione. Second, the eluted GST protein is
run over a Nap10 column to remove the glutathione, resulting in a very pure sample
containing only the GST fusion protein.
REFERENCE
Austin, B. P., Nallamsetty, S., & Waugh, D. S. (2009). Hexahistidine-tagged maltose-
binding protein as a fusion partner for the production of soluble recombinant
proteins in Escherichia coli. Methods Mol Biol, 498, 157-172. doi:10.1007/978-
1-59745-196-3_11
BioLabs. (2016). Maltose Binding Protein Expression. Retrieved from
https://international.neb.com/applications/protein-expression-and-
purification/coupled-protein-expression-and-purification/maltose-binding-
protein-expression
Needle, D., & Waugh, D. S. (2014). Rescuing aggregation-prone proteins in
Escherichia coli with a dual His(6)-MBP tag. Methods Mol Biol, 1177, 81-94.
doi:10.1007/978-1-4939-1034-2_7
Simons, P. C. a. V., D.L. (1977). Purification of glutathione S-transferases for human
liver by glutathione-affinity chromatography (Vol. 82:334-41): Anal Biochem.
ThermoFIsher. (2014). GST-tagged Proteins – Production and Purification. Retrieved
from https://www.thermofisher.com/id/en/home/life-science/protein-
biology/protein-biology-learning-center/protein-biology-resource-
library/pierce-protein-methods/gst-tagged-proteins-production-
purification.html
Zhao, X., Li, G., & Liang, S. (2013). Several Affinity Tags Commonly Used in
Chromatographic Purification. Journal of Analytical Methods in Chemistry,
2013, 8. doi:10.1155/2013/581093