Protein Purification: Isolation, Characterization and Quantification of β-
galactosidase
A project about isolation, characterization, and quantification of His-tag labeled β-galactosidase
Valerie van den Heuvel
10th January 2018
Abstract
Protein purification constitutes very important steps during downstream processing of therapeutic
proteins. Contaminants in the final formulation of a product may lead to the activation of the immune
system of the patient. Protein purification may be performed by different techniques including filtration,
centrifugation, and a various range of analytical techniques. Purification may be facilitated by the use
of covalently linked tags in which the purification is based on affinity. This final report aims at the
isolation and characterization of His-tag labeled β-galactosidase using Ni-NTA resin and SDS-PAGE.
Furthermore, the project was aimed at the quantification of the enzymatic activity and concentration
following the release of ortho-nitrophenyl-β-galactoside, and the Bradford assay respectively.
Purification using Ni-NTA resin according to the batch method showed that the used purification
method was not sufficient for obtaining high purity β-galactosidase. During this project, the principle of
the quantification of the enzymatic activity and concentration was determined but further research
should be performed in order to obtain more significant reliable results.
, Protein Purification: Isolation, Characterization, and Quantification of β-galactosidase | Valerie van den Heuvel
1. Introduction proteins to ‘stack’ into a tight band before entering
the running gel. This allows proteins to start
β-galactosidase is an important type of running at the same time since the technique
glycoside hydrolase enzyme that catalyze the separates molecules by their size. In the running
conversion of lactose to glucose and galactose.1–4 gel, a higher percentage crosslinking of the
The enzyme specifically attacks β-galactosides via polyacrylamide is found to achieve higher
intermediate formation of a covalent bond. The resolution (smaller bands).9
primary natural substrate is lactose which is either Furthermore, a pH gradient is applied during
converted tot galactose and glucose (hydrolysis) the experiment, ranging from 6.8 in the top to 8.8
or allolactose (galactosyl transfer).5 During the at the bottom. The Tris-HCl buffer in the tank
reaction, the participation of two acidic amino acid contains glycine which can exist in three different
residues is required. In general, one amino acid charge states depending on the pH. At a pH of 6.8,
residue serves as an acid/base and the other as a glycine is present in zwitterionic (naturally
nucleophile that polarizes the water molecule charged) state. The naturally charged state results
targeting the glycosyl-enzyme intermediate.2,5,6 in slow migration in the electric field. On the other
hand, the Cl- ions (from Tris-HCl) move with a
During the production of a therapeutic protein, higher in the electric field towards the anode
the incorporation of purification steps is essential. resulting in the formation of an ion that migrates
In industrial applications, a suitable tag can be ahead of the glycine. When the pH switches to 8.8,
covalently linked to the protein to facilitate the most glycine molecules are negatively charged
purification process in which separation is and migrate much faster than the proteins towards
achieved based on affinity.7 During this project, the anode. The glycine front accelerates past the
His-tag labeled β-galactosidase was purified from proteins, leaving them behind resulting in a
a Rosetta-gami B lacZ lysate by the use of HisPur narrow band in the interface of the stacking and
Ni-nitrilotriacetic acid (NTA) resin. This resin running gel since the running gel has an increased
binds specifically to His tag labeled substrates acrylamide concentration. This slows the
containing 6 histidine residues (in this case β- movement of the proteins according to their size
galactosidase). During the experiment several and separation begins.10
centrifuge steps are incorporated to remove
contaminants present in the original lysate. The Finally, for a manufacturer it is important to
different buffers used contain increasing know the concentration of the protein of interest in
imidazole concentrations. During the purification the suspected fraction and its activity. By the use
experiment, the imidazole containing buffers of a spectrophotometric assay, the activity of β-
function as a competitive agent to eventually elute galactosidase in the different fractions can be
the protein of interest from the HisPur Ni-NTA determined. The enzymatic activity of β-
resin.8 galactosidase can be quantified following the
release of ortho-nitrophenyl-β-galactoside
After separation of the protein of interest from (ONPG) at 420 nm.11 ONPG, structurally similar
the lysate, a suitable separation technique needs to to lactose, consists of a beta-D-galactoside
be performed in order to determine the presence of substituted with a 2-nitrophenyl substituent at the
the protein of interest in the suspected fraction. anomeric position (lactose has a glucose
This can be visualized by sodium dodecyl sulfate- substituent).12 Under normal circumstances
polyacrylamide gel electrophoresis (SDS-PAGE), ONPG is a colorless substrate. Upon hydrolysis in
an analytical technique used for the separation of the presence of β-galactosidase, ONPG cleaves
molecules based on differences in molecular into two residues, galactose and o-nitrophenol
weight. In this technique, SDS serves as a
(Figure 1). The o-nitrophenol residue exhibits a
detergent that denatures secondary and
yellow color and can therefore be used for the
nondisulfide-linked tertiary structures.
determination of the enzymatic activity based
Additionally, SDS coats the molecules with a
color intensity (at 420 nm).13
strong negative charge correlating with their
length, allowing molecular weights to be
Although many advantages of this method
estimated. The classical SDS-PAGE system is a
regarding its simplicity, there are some important
discontinuous gel consisting of an upper stacking
things to consider. First of all, the color intensity
gel and a lower running gel which have different
of ONPG is pH dependent. The use of a high pH
pH values and polyacrylamide concentration
(around 8.0) results in deprotonation of the
(regarding cross-linking). The stacking gel has a
hydroxyl group and the strongest yellow color will
lower percentage of polyacrylamide allowing
appear. However, such pH may inhibit the enzyme
Page 1/8
galactosidase
A project about isolation, characterization, and quantification of His-tag labeled β-galactosidase
Valerie van den Heuvel
10th January 2018
Abstract
Protein purification constitutes very important steps during downstream processing of therapeutic
proteins. Contaminants in the final formulation of a product may lead to the activation of the immune
system of the patient. Protein purification may be performed by different techniques including filtration,
centrifugation, and a various range of analytical techniques. Purification may be facilitated by the use
of covalently linked tags in which the purification is based on affinity. This final report aims at the
isolation and characterization of His-tag labeled β-galactosidase using Ni-NTA resin and SDS-PAGE.
Furthermore, the project was aimed at the quantification of the enzymatic activity and concentration
following the release of ortho-nitrophenyl-β-galactoside, and the Bradford assay respectively.
Purification using Ni-NTA resin according to the batch method showed that the used purification
method was not sufficient for obtaining high purity β-galactosidase. During this project, the principle of
the quantification of the enzymatic activity and concentration was determined but further research
should be performed in order to obtain more significant reliable results.
, Protein Purification: Isolation, Characterization, and Quantification of β-galactosidase | Valerie van den Heuvel
1. Introduction proteins to ‘stack’ into a tight band before entering
the running gel. This allows proteins to start
β-galactosidase is an important type of running at the same time since the technique
glycoside hydrolase enzyme that catalyze the separates molecules by their size. In the running
conversion of lactose to glucose and galactose.1–4 gel, a higher percentage crosslinking of the
The enzyme specifically attacks β-galactosides via polyacrylamide is found to achieve higher
intermediate formation of a covalent bond. The resolution (smaller bands).9
primary natural substrate is lactose which is either Furthermore, a pH gradient is applied during
converted tot galactose and glucose (hydrolysis) the experiment, ranging from 6.8 in the top to 8.8
or allolactose (galactosyl transfer).5 During the at the bottom. The Tris-HCl buffer in the tank
reaction, the participation of two acidic amino acid contains glycine which can exist in three different
residues is required. In general, one amino acid charge states depending on the pH. At a pH of 6.8,
residue serves as an acid/base and the other as a glycine is present in zwitterionic (naturally
nucleophile that polarizes the water molecule charged) state. The naturally charged state results
targeting the glycosyl-enzyme intermediate.2,5,6 in slow migration in the electric field. On the other
hand, the Cl- ions (from Tris-HCl) move with a
During the production of a therapeutic protein, higher in the electric field towards the anode
the incorporation of purification steps is essential. resulting in the formation of an ion that migrates
In industrial applications, a suitable tag can be ahead of the glycine. When the pH switches to 8.8,
covalently linked to the protein to facilitate the most glycine molecules are negatively charged
purification process in which separation is and migrate much faster than the proteins towards
achieved based on affinity.7 During this project, the anode. The glycine front accelerates past the
His-tag labeled β-galactosidase was purified from proteins, leaving them behind resulting in a
a Rosetta-gami B lacZ lysate by the use of HisPur narrow band in the interface of the stacking and
Ni-nitrilotriacetic acid (NTA) resin. This resin running gel since the running gel has an increased
binds specifically to His tag labeled substrates acrylamide concentration. This slows the
containing 6 histidine residues (in this case β- movement of the proteins according to their size
galactosidase). During the experiment several and separation begins.10
centrifuge steps are incorporated to remove
contaminants present in the original lysate. The Finally, for a manufacturer it is important to
different buffers used contain increasing know the concentration of the protein of interest in
imidazole concentrations. During the purification the suspected fraction and its activity. By the use
experiment, the imidazole containing buffers of a spectrophotometric assay, the activity of β-
function as a competitive agent to eventually elute galactosidase in the different fractions can be
the protein of interest from the HisPur Ni-NTA determined. The enzymatic activity of β-
resin.8 galactosidase can be quantified following the
release of ortho-nitrophenyl-β-galactoside
After separation of the protein of interest from (ONPG) at 420 nm.11 ONPG, structurally similar
the lysate, a suitable separation technique needs to to lactose, consists of a beta-D-galactoside
be performed in order to determine the presence of substituted with a 2-nitrophenyl substituent at the
the protein of interest in the suspected fraction. anomeric position (lactose has a glucose
This can be visualized by sodium dodecyl sulfate- substituent).12 Under normal circumstances
polyacrylamide gel electrophoresis (SDS-PAGE), ONPG is a colorless substrate. Upon hydrolysis in
an analytical technique used for the separation of the presence of β-galactosidase, ONPG cleaves
molecules based on differences in molecular into two residues, galactose and o-nitrophenol
weight. In this technique, SDS serves as a
(Figure 1). The o-nitrophenol residue exhibits a
detergent that denatures secondary and
yellow color and can therefore be used for the
nondisulfide-linked tertiary structures.
determination of the enzymatic activity based
Additionally, SDS coats the molecules with a
color intensity (at 420 nm).13
strong negative charge correlating with their
length, allowing molecular weights to be
Although many advantages of this method
estimated. The classical SDS-PAGE system is a
regarding its simplicity, there are some important
discontinuous gel consisting of an upper stacking
things to consider. First of all, the color intensity
gel and a lower running gel which have different
of ONPG is pH dependent. The use of a high pH
pH values and polyacrylamide concentration
(around 8.0) results in deprotonation of the
(regarding cross-linking). The stacking gel has a
hydroxyl group and the strongest yellow color will
lower percentage of polyacrylamide allowing
appear. However, such pH may inhibit the enzyme
Page 1/8
