1. ABSTRACT
MATERIALS & METHODS
RESULTS
Purification of Beta-galactosidase from E. coli
B. Eccleston, B. Acharya, J. Cox , B. Curry, K. Killam, S. King, N. Richardson, J. Yang, W. Briscoe, PhD.
Tulsa Community College Department of Biotechnology
CONCLUSIONS AND FUTURE DIRECTIONS
REFERENCES
ACKNOWLEDGEMENTS
The purpose of our study was to isolate and purify the enzyme Beta-Galactosidase. In doing this,
we were able to explore and understand the different processes involved in protein purification. E. coli
15224, a mutant strain that produces B-gal constitutively, was grown in nutrient broth and centrifuged
to a pellet. The pelleted cells were then lysed by sonication and centrifuged to pellet out cell debris
while the majority of proteins remained in the crude lysate. Bradford assays were performed to
determine protein concentration and ONPG assays were used to detect b-gal activity. In order to
perform ammonium sulfate precipitation, we had to determine a suitable percentage of saturation that
would precipitate B-gal. After the proper AmmSO4 was determined, we performed dialysis which
desalted our sample. The samples were then purified by Ion-Exchange Chromatography. After Ion-
Exchange, the samples were then further purified by Affinity Chromatography. Archived samples
from each purification step were then ran on an SDS-PAGE for analysis.
Specific Activity at Each Purification
Avg B-
gal Avg Bradford Specific Activity
Crude lysate 24.72 2.54 9.73
0-30% 2.41 0 0
30-45% 23.93 0.36 66.47
45%+ 6.49 1.53 4.24
IEC3 23.86 0.42 56.81
IEC4 24.81 0.43 57.70
AFFc 1 11.31 0 0
AFFc 2 19.54 0.12 162.83
Husain, 2010, Beta galactosidases and their potential applications: a review, Critical
Reviews in Biotechnology, (1):41-62
Lodish, Harvey, David Baltimore, Arnold Berk, S. Lawrence. Zipursky, Paul Matsudaira,
and James Darnell. Molecular Cell Biology. New York: Scientific American, 1995. Print.
"Enzyme Manual: Galactosidase, Beta." Enzyme Manual: Galactosidase, Beta. Worthington
Biochemical Corporation, 2016. Web. 28 Apr. 2016.
Boyer, Rodney F. Modern Experimental Biochemistry. San Francisco: Benjamin Cummings,
2000. Print.
PerfectProtein15-150kDa
Crudelysate
0-30%AS
30-45%AS
45+%AS
IEC3
IEC4
AffC1
AffC2
SigmaB-Gal
AffC1
BioRadKaledescoprStd
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 20 40 60 80
Absorbance(AU)
Tube
Ion- Exchange Chromatography Analysis
BA/BE bradford
BC/JY bradford
KK/ Bgal
NR Bgal
Figure 1. Ion-Exchange Chromatography using 30-45% AS
on a gradient of 0.25M to 0.75M NTM
0
0.5
1
1.5
2
2.5
3
3.5
0 20 40 60 80
Absorbance(AU)
Tube
Affinity Chromatography Analysis
Bradford
B-Gal
Figure 2. Affinity Chromatography of fractions 26-38 from IEC 4
Figure 3. Specific Activity at each purification step.
Figure 4. SDS-PAGE visualization of each purification step.
Dialysis or
Centrifugal
Filtration
Ion-Exchange
Chromatography
Affinity
Chromatography
SDS-PAGE Gel
Testing for proper Ammonium Sulfate percentages for prec. of B-gal:
Purification steps for the re-suspended pellet of 30-45% AmmSo4 :
E. coli 15224 cells were cultured and harvested for sonication in breaking buffer followed by
centrifugation. After centrifugation, the pelleted cells were resuspended in Z-buffer.
All pellets and
supernatants were
assayed for specific
activity. The pellet from
the 30-45% was chosen
for further purification
due to its high specific
activity of Beta-
Galactosidase.
Buffers
Phosphate buffer
Breaking buffer
Z-buffer
5.7ml crude
lysate +
1.123g
AmmSO4 =
0-35%
5.95ml
supernatant +
0.524g
AmmSO4 =
35-50%
5.85ml
supernatant +
0.742g
AmmSO4 =
50-70%
Supernatant
70+%
Pellet
Centrifuge Centrifuge Centrifuge
PelletPellet
Centrifuge Centrifuge
0-30%
AmmSO4
30-45%
AmmSO4
Supernatant
45+%
AmmSO4
Supernatant
PelletPellet
The pellet from the 35-50% AmmSo4 fraction had the highest specific
activity from the above diagram. The diagram below shows AmmSo4%
testing within a narrow range between 0-45+%.
Assay reagents
1X Bradford dye
ONPG (4mg/ml)
1M Sodium
Carbonate
INTRODUCTION
The ability to produce and purify proteins from genetically modified organisms has been an
important aspect of biotechnology since the advancement of DNA science. A protein of interest
can be purified from cultured prokaryotic or eukaryotic cells that have been genetically engineered
to express a gene constitutively. Several techniques can be utilized for the separation and
purification of the target protein including sonication, centrifugation, ammonium sulfate
precipitation, dialysis, ion exchange chromatography, affinity chromatography, and SDS-PAGE.
Samples between each purification step are assayed to measure protein concentration (mg/ml) and
enzyme activity (units/ml) in order to determine specific activity. Specific activity is a
measurement of protein purity, and is defined as enzyme activity (units/ml) over total protein
concentration (mg/ml). Enzymes are crucial in many biological activities including but not limited
to cell structure, transport, and chemical reactions crucial to energy production. The enzyme beta-
galactosidase (b-gal) is a 464-kDa tetramer transcribed from the lac operon gene which converts
lactose into glucose and galactose. B-gal can be used to remove lactose from milk for people who
are lactose intolerant. B-gal can also be applied to the production of other galactosylated products.
ONPG
assay
Bradford
assay
Column Chromatography
• According to results, purification of beta-galactosidase from E. coli is best accomplished by
a combination of techniques.
• Cell lysis by sonication was followed by purifying the samples, based on solubility, by
means of 30-45% ammonium sulfate precipitation.
• Ion exchange chromatography was found to have the most success with a 0.25→0.75
molarity NTM gradient.
• Further product purification was accomplished by using affinity chromatography with p-
aminobenzyl-1-thio-B-D-galactopyranoside.
• After each step, Bradford and ONPG assays were performed to monitor specific activity of
B-gal.
• After completion of these techniques, the final specific activity was 159 units/mg. SDS-
PAGE verified the isolation and purity of B-gal with one band at ≈ 120 kDa. This monomer
corresponds to the known molecular weight of each identical subunit of the B-gal tetramer.
• It was found that increasing the concentration of sample in the Bradford Assay led to more
accurate estimates of protein concentration and thus specific activity.
• Decreasing the concentration of sample in the ONPG Assay would lead to more accurate
estimates of B-gal.
We would like to acknowledge Tulsa Community College for use of their facilities and
great faculty members. We would also like to thank INBRE and NSF for the funding that
made this project possible. Finally, we would like to thank Dr. William Briscoe for his
guidance and training in the techniques necessary to accomplish this research.