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Report for the course biological drugs at Utrecht university
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The examination of Beta galactosidase purification process by determining specific activityand performing SDS page analysis
Author: Donya Jadidi Fighan
Student number: 6738729
Date of report: 19/06/2022
,Introduction
β-Galactosidase is an enzyme that is encoded by the gene lacZ in operon lac. Not only is it
capable of hydrolysing lactose to glucose and galactose, but also it catalyses the conversion
of lactose to allolactose. An important characteristic of this enzyme is that it only can be
expressed in presence of lactose. By the time lactose has been degraded, β-Galactosidase
inactivates.[1] In this study, this enzyme is produced by Bl21-pIVEXLacZHis6, which is
derived from the transformation process of BL21 E. coli with pIVEXLacZHis6 plasmid. The
plasmid contains a His-tag leading to producing His6-galactosidase that can be purified by
affinity chromatography. This is due to the high affinity of histidine to nickel ions present in
the column. In order to induce the activation of β-Galactosidase, isopropyl beta-D-
thiogalactoside (IPTG) is used, by which, the constant activation of target enzyme will be
achieved as this substance will not be degraded as apposed to lactose.[2] The aim is to
purify β-Galactosidase from the resulted lysate after the upstream processing and examine
weather the purification process was efficient by determining the activation and concentration
of this enzyme followed by SDS page analysis.
On the first day of experiment, purification was done by affinity chromatography. Bacterial
lysate containing the target protein (β-Galactosidase) was already provided. To purify the
enzyme an increasing concentration of imidazole was used during this process since this
substance has also the affinity to bind to the nickel ions of the column, thereby competing
with histidine. At the end, fractions with high concentration of target enzyme were collected
for the next experiments.
Following the purification, the activation of β-Galactosidase was determined by ortho-
Nitrophenyl-β-galactoside (ONPG), which is a colourless substance. However, after reacting
with β-Galactosidase, a yellow product called ortho-nitrophenol (ONP) is produced that is a
measure for the enzyme activity. By a spectrophotometer, β-galactosidase activity is
quantified at 420 nm to realize the amount of substrate that is converted at this
wavelength.[3] To be able to determine the activity of the enzyme, a calibration curve was
made by known activations of β-Galactosidase as the standards.
The third experiment is measuring the concentration by applying Bradford assay. This
method is not capable of detecting different proteins. Therefore, the concentration of all
proteins will be measured in this experiment and not only β-Galactosidase. This method is
based on Coomassie brilliant blue G250, which is a dye showing different colours at different
PHs. In an acidic environment it represents a reddish or brownish colour, while after binding
to the protein, it turns blue. The dye interacts with arginine, lysine and histidine since its
negative sulfonic acid group interacts with the positive amino groups of these basic amino
acids. This leads to shifting the maximum absorption from 470 nm to 595 nm. The
spectrophotometer measures the optical density of the sample at 595nm. Having made the
calibration curve by known concentrations of BSA (Bovine serum albumin), the concentration
of the protein can be calculated in our unknown solutions.[4]
The last experiment was SDS-page (SDS polyacrylamide-gel electrophoresis), which
separates proteins based on their size and molecular mass. Polyacrylamide gel is used as a
matrix to help the migration of proteins. Proteins are first dissolved in a solution containing
sodium dodecyl sulphate (SDS) which carries a negative charge and has a long carbon
chain. SDS is capable of binding to the hydrophobic regions of proteins, thereby unfolding
them. Moreover, to break down the S-S bonds a reducing agent such as β-mercaptoethanol
is normally added so that all the polypeptides can migrate separately. When voltage is
applied, the proteins migrate to the positive electrode as the negative charge of SDS covers
, the intrinsic charge of proteins. This latest argument leads to separating the proteins based
on their size so the smaller a protein, the more quickly it moves.[5]
Methods and materials
• β-galactosidase protein purification
As stated in the manual; having shaken the HisPur Ni-NTA, 500 micro litre of the suspension
was centrifuged for 2 minutes at 700xg in an Eppendorf tupe of 0,2 mL and the supernatant
was removed. When 1 ml equilibration-buffer (20 mM sodium phosphate, 0.3 M sodium
chloride, 10 mM imidazole; pH 7.4) was added and shook, the suspension was centrifuged
for another 2 minutes, and the supernatant was also removed. Following that, 750 µL lysate
was mixed with 750 µL equilibration buffer which was added to the pellet of previous step
and mixed for 30 minutes. Next, the sample was centrifuged, and the supernatant was
harvested for further analysis in an 1,5 mL ep. Then, the pellet was washed by 1000 µL wash
water (20 mM sodium phosphate, 300 mM sodium chloride, 25 mM imidazole; pH 7.4)
followed by centrifuging at 700xg for 2 minutes, after which the supernatant was harvested
and stored in an 1,5 mL Eppendorf tube. (wash1) This step repeated for another two times to
make the wash 2 and 3 fractions. Lastly, 250 μL elution buffer (20 mM sodium phosphate,
0.3M sodium chloride, 0.2M imidazole; pH 7.4) was added to the resin leading to the elusion
of bounded protein, after which this step was performed for another 2 times and the
supernatant was stored in separate 1,5 Eppendorf tubes. All these fractions were stored at
20 °C, before which 30 % glycerol was added to them in order to reach an end concentration
of 10% glycerol. The fractions were used for the rest of the experiments. [6]
• β-galactosidase activity
First of all, with 100 U/ml β-galactosidase stock solution the dilution series of known activities
(0.1-3.0 units/ml) were made so as to obtain a calibration curve. Instead of water, glycerol
10% was used to perform the dilutions. Moreover, undiluted, 10x diluted and 100 times
diluted samples were also prepared to determine their activity. Note that all the dilutions were
made two times inclusive the blank which was glycerol 10% (duplicates). Secondly, 15 µL of
standards and samples were added to specific parts of the 96-Well Microplates. Thirdly, 60
µL Z-Buffer (60 mM Na2HPO4 , 40 mM NaH2PO4 , 10 mM KCl, 1 mM MgCl2, 1 mM
Dithiothreitol (DTT)) was added to each of them, after which they were all incubated for 5
minutes at 37° C. (manual says 10 minutes) Then the reaction was induced by adding 60 µL
ONPG followed by incubating for 15 minutes at 37° C. Finally, 120 µL of the stop solution (1
M Na2CO3) was added. Having reached the room temperature, the value of activity was
determined at 420 nm. The values were used to draw the calibration curve.[6]
• β-galactosidase protein concentration
First of all, the dilution series, which had a range of 0 to 100 µg/ml BSA were made by
diluting the BSA stock solution(2mg/ml) in duplicates. In addition, the samples were also
prepared as undiluted and 5x diluted to make sure that they are within the range of
calibration series. Having pipetted 20 µL of each standard series, the diluted and undiluted
samples into the 96-Well Microplates, 180 µL of the Bradford dye (Bio-Rad 500-0006) was
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