The document summarizes experiments conducted to express and isolate the ALDH1A1 protein using bacterial vectors. E. coli containing the ALDH1A1 gene were cultured and lysed using sonication. Size exclusion chromatography was used to separate the protein from cell debris, followed by affinity chromatography to further purify the protein. Enzyme activity was confirmed through kinetic assays and the purified protein was analyzed using SDS-PAGE, Western blot, dot blot, and ELISA, confirming isolation of the 50kDa ALDH1A1 protein band. Troubleshooting of techniques like sonication was required but the overall process was successful in expressing and separating the ALDH1A1 protein.

1. ALDH1A1 PROTEIN
EXPRESSION WITH
BACTERIAL VECTOR II
By Jason Morris and Antony Crane of Minneapolis
Community and Technical College, 1501 Hennepin
Avenue, Minneapolis, MN, 55403
ABSTRACT
This article is a continuation of a series of experiments in
which pET-Blue expression vector containing ALDH1A1
gene was transfected into E. coli BL21DE3pLysS and
selectively confirmed via antibiotic resistance on LB-agar
plates. Colonies with resistance were selected for scale-up
broth and incubated no longer than 20 hours without
inducing agent iPTG for increased mitotic division. Scale up
process started by adding inducing agent iPTG after desired
optical density (OD) was achieved via spectrophotometric
analysis to produce maximum ALDH1A1. Intracellular
protein pellet was later kept in lysis buffer with EDTA and
PMSF at cryogenic temperatures. This protein expression
experiment was continued by checking enzyme activity of
each pair of technicians’ proteins out of cryogenic freeze,
sonicating, and centrifuging cell debris. The supernatant
containing ALDH1A1 was kept for size exclusion (gel
filtration) chromatography to be followed by Affinity
Chromatography with a nickel column stationary phase to
further isolate the protein of interest ALDH1A1 from
interfering impurity proteins. Samples from various points
of chromatographic separation were ran on polyacrylamide
gel electrophoresis (PAGE). Finally a Western blot, dot blot,
and enzyme-linked immunosorbent assay (ELISA) were
done on ALDH1A1. The sonication step needed
troubleshooting for some technicians but was mainly a
success.The enzyme activity was promising for most
technicians, but inactive for others. The Western,dot, and
ELISA showed that the chromatographic procedures indeed
separated the ALDH1A1 protein as desired with a 50kDa
band.
Conducted April 7th-April 27th, 2015
In partial fulfillment of Biochemistry Laboratory
and Techniques Class Project under the
supervision of Dr. Rekha Ganaganur, spring
semester, 2015

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Introduction:
The second half, or downstream process, of the ALDH1A1 project involved separation of the
ALDH1A1 protein from the cells and verifying the protein’s expression while keeping the protein active.
Since our protein occurred in the cytosol of the cell, we didn’t need to use a non-ionic detergent to
remove the protein from a membrane. We instead used a sonicator to lyse the cells and followed that with
high speed centrifugation, using only the supernatant fluid for our continued protein separation.
We then applied size exclusion chromatography to the supernatant to isolate our protein from
most of the other impurities caused by the lysis, followed by affinity chromatography through a charged
nickel column to further separate our protein from additional impurities. Along the way, to make sure we
had at least an active protein present, using Bradford reagent we tested samples of eluates with various
buffers, verifying an active protein in all our samples by the presence of a blue color. We also verified an
active version of ALDH1A1 by using a kinetic assay of our samples and running those through a
spectrophotometer immediately after the addition of a compatible substrate (acetaldehyde).
Following the verification of our enzyme kinetics through the spectrophotometer, we received
further confirmation of ALDH1A1 through an SDS-PAGE gel, Western blot, dot blot and ELISA assay.
The SDS-PAGE showed some unexpected impurities present. However,those impurities can probably be
removed via further affinity chromatography.

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Materials and Methods:
Enzyme activity (bioactivity) was verified with a spectrophotometer after thawing cells.
Sonication cycles were carried out at 6 minute cycles divided into 12 total bursts with 30 second cooling
intervals between each burst to avoid destroying enzyme activity. Bursts were carried out for 20 seconds
each at 10% power amplitude. Lysate centrifuged at 40,000xG at 4 °C for 45 minutes. Removal of lysis
debris impurities by gel filtration chromatography was done with a SephadexR
G-25 resin. Kelly Stedman,
CLA hydrated, swelled, and degassed the resin. The column was packed with 8mL 0.15M NaCl and
8.5mL of the slurry. 2 column volumes of 0.1M NaCl were passed through along with 15mL of
equilibration B.
Affinity chromatographic method was used with Nickel coated stationary phase to bind His-
tagged ALDH1A1 and allow impurities to pass through. Some weak nonspecific proteins may bind but
will elute out first (1). Imidazole containing buffer was therefore used in increasing concentrations to
elute out first impurities, and eventually the His-tagged ALDH1A1. Imidazole acts as a competitive
ligand. Samples of eluate were collected not only from each step of affinity chromatography but gel
filtration as well, and each of these sample fractions were tested via Bradford assay to determine how
much/if any protein were present in each fraction based on the intensity of blue color remained. It was
determined that the majority of ALDH1A1 protein was eluted with 250mM imidazole, as compared to 25
and 60mM. Another pass of 500mM imidazole was also collected just in case there was leftover
ALDH1A1 in the column. Column had Nickel removed with EDTA buffer and salts solution to remove
EDTA.
Kinetic assay involving reduction of NAD+
to NADH at 340nm (UV-A) was done using a crystal
quartz cuvette. GSH, pyrazole, ALDH1A1,NAD,sodium pyrophosphate buffer, and substrate
acetaldehyde were all required in varying concentrations for the reaction. Polyacrylamide gel
electrophoresis (40% stock acrylamide) done with sample fractions leftover from chromatographic
separation. Running gel buffer was 1.5M Tris-HCl at basic pH of 8.3. Refer to protocol, results section,
and conclusions for further details of Western blot, dot blot, and ELISA procedures.
To ensure many of the instruments were working properly, routine maintenance was performed.
Such as a routine examination of the steam autoclave, or general cleaning and inspection of the
micropipettor sets. Expiration dates were consistently examined for reagents and various materials. The
4 °C cold room is equipped with temperature sensors and the entire cold room is modified at a certain
negative pressure with alarms equipped in case of fluctuations outside the allowable threshold.
Instruments used in these experiments are listed below.

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of May, 2015 ALDH1A1 Protein Expression Page 3 of 13
Various technical procedures conducted with the following instruments:
-FisherScientificSonicDismembrator;Model 500;
Serial #BCKO6082J04; RoomS3600; Lot # BIOT-0165
-BECKMAN COULTER AllegraCentrifuge;Model 64R;
Serial # ALV08D06; ID # BIOT-CN03
-Mini-PROTEAN TGXPolyacrylamideGel byBIORAD;
Exp 2015-12-11; Catalog# 456-1094
-VWROrbital Shaker;ID # BIOT-0101; Serial # 41694
-CPSController;ID# BIOT-0128; Serial #
A1039430483TK; RoomS3300
- Bio-RadLabsSmartSpecPlusSpectrophotometer;
Serial # 273 BR 07396
- BeckmanCoulterAllegraX-22R Centrifuge;
Serial # ALB03F019; ID # BIOT-0118
-Eppendorf Centrifuge;Model 5415D; Serial # 5425-
26984; ID # BIOT-0115
-IsotempFisherScientific;Model 2052FS Dryblock
heater;Serial #1649080344320

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of May, 2015 ALDH1A1 Protein Expression Page 4 of 13
Results:
Sonication:
High speed centrifugation:
At 40,000 Gs applied for 45 minutes, we received a hefty size pellet and mildly turbid supernatant fluid.
Used: Beckman Coulter Allegra 64R Centrifuge (s/n ALV08D06)
Size exclusion chromatography:
Had three fractions come out of our chromatography. The first tube, ran
through with 2.5 mL lysate, had a slight yellow tint to it. The second
tube, ran through with 2.5 mL of buffer B, had a yellow tint two shades
darker than the first tube. The third tube, ran though with 2.5 mL buffer
B, was mostly clear. Applying 50 uL of each sample to a 96 well plate
and then adding 150 uL of Bradford reagent revealed a blue color, and
therefore a protein, within each tube, although the tube that should have
our target protein is the second tube.
Performed the sonication twice, using 4 total minutes of
sonication each time. The main reason the second
application of sonication was required was due to the
awkwardness of getting the “feel” correct for the position
of the sonic dismembrator in the sample. Didn’t think it
was applied correctly the first time so a second time was
implemented to make sure it was done correctly.
Used: Sonifer Sound Enclosure (s/n VSW309208014-
075)
Sonifer Control Unit (s/n BCK06082504)
Sonic dismembrator (s/n QBV06075000)Pictured: Sonicator with sample being sonicated.
Pictured: Size Exclusion column

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Affinity chromatography:
Nickel column set up was slower than most of the columns in class due to math errors in reading the
column height. However,may have given a greater volume than others in terms of the results received,
particularly at later stages.
Using flowthrough from the second tube from size exclusion chromatography as the source for our
affinity chromatography, achieved the following fractions:
Buffer B: similar tint of yellow as the size exclusion
chromatography tube, positive for protein with Bradley reagent
Buffer C1: completely clear, positive for protein with Bradley
reagent
Buffer C2: completely clear, positive for protein with Bradley
reagent
Buffer C3: completely clear, positive for protein with Bradley
reagent
Buffer D: completely clear, positive for protein with Bradley
reagent, darker blue than previous samples.
Enzyme kinetics:
Used 100 uL sample from Buffer C3 and 100 uL from our size exclusion buffer and combined separately
with:
500 uL water,100 uL NAD (80 mM stock), 100 uL GSH (100 mM stock), 100 uL pyrazole (2 mM
stock), 1 mL sodium pyrophosphate buffer (64 mM stock and 2 mM EDTA). Added substrate at last
moment before running the assay through spectrophotometer.
Incubated in 37ºC dry heater before running the kinetic assay in Shimadzu UV-2450 Spectrophotometer
(s/n A10834338005CS)
Pictured: Buffer C3 and Buffer B

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of May, 2015 ALDH1A1 Protein Expression Page 6 of 13
Our results:
SDS-PAGE gel:
1 2 3 4 5 6 7 8 9 10
Lane 1: marker loaded with 15 uL
Lane 2: Uninduced IPTGwith 38 uL
Lane 3: Induced IPTGwith 38 uL
Top gray line:
size exclusion sample
Top blue line:
Buffer C3 sample
Pictured: graph of
results from several
teams’ samples.
Pictured: shows SDS-PAGE
gel with different lanes
corresponding to different
fractions gathered at various
steps in the downstream
process of analysis of our
ALDH1A1 protein.
Results were as anticipated. Take
notice of the heavy impurities from
lanes 2 through 5 as expected, and the
thick, pure ALDH1A1 band on lane 8
with minimal impurities. It is
approximately 50kDa which BSA
protein standard in lane 10 is also
approximately 50kDa as well.

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of May, 2015 ALDH1A1 Protein Expression Page 7 of 13
Lane 4: Before size exclusion with 38 uL
Lane 5: Post size exclusion (buffer B) with 38 uL
Lane 6: C1 buffer with 38 uL
Lane 7: C2 buffer with 38 uL
Lane 8: C3 buffer with 38 uL
Lane 9: D buffer with 38 uL
Lane 10: BSA protein standard with 38 uL
Western Blot:
1 2 3 4 5 6 7 8 9 10 Western blot results were similar to that
of SDS-PAGE as expected. The 50kb
BSA protein standard however seems to
have shown up on lane 5 with unknown
origins of cause instead of lane 10.
Lane 1: marker loaded with 15 uL
Lane 2: Uninduced IPTGwith 38 uL
Lane 3: Induced IPTGwith 38 uL
Lane 4: Before size exclusion with 38 uL
Lane 5: Post size exclusion (buffer B)
with 38 uL
Lane 6: C1 buffer with 38 uL
Lane 7: C2 buffer with 38 uL
Lane 8: C3 buffer with 38 uL
Lane 9: D buffer with 38 uL
Lane 10: BSA protein standard with
38 uL
Pictured: Western Blot

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of May, 2015 ALDH1A1 Protein Expression Page 8 of 13
Dot Blot:
The results were well considering
what technician Gregg managed to
do. Even though the entire
secondary antibody was removed
shortly after exposure to it, presence
of the antibody for ALDH1A1 still
showed up. This is likely because
CLA Kelly Stedman had a small
amount of backup secondary
antibody available.
ELISA:
ELISA results contains a different
class group of two students’ data
for each column. Each column
thus has the same samples,
however each individual column
does contain more or less ALDH
protein. Each row was assigned a
different substrate. Row 1 had 5-
Bromo-4-chloro-3-indolyl
phosphate (BCIP),row 2 had
paranitrophenyl phosphate (PNPP),
and row 3 had tetra methyl
benzidine (TMB). Each color
showed up for each substrate as
expected with the exception of
severalstudent groups for
substrate BCIP.
Discussions:
Pictured: Dot Blot
Pictured: 1st and 3rd rows of ELISA
Pictured: 1st and 2nd rows of ELISA
Top middle dot contains data analyzed.

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of May, 2015 ALDH1A1 Protein Expression Page 9 of 13
It was expected that each step would further the isolation of ALDH1A1 from other impurities
surrounding the protein. Despite initial concern that the vortexing done earlier would denature the
ALDH1A1,it seemed, especially in comparison with other classmates,that our ALDH1A1 fared better
than expected. While the sonication was going on, we had to wear ear protection and make sure we didn’t
touch the tube with the sonicator in our initial setup to prevent shattering of both our eardrums and the
tube.
The second elution step of the size exclusion chromatography was expected to have the enzyme,
while the first and third step were not. The reasoning for this is that the ALDH1A1 molecules would be
initially trapped with the first step while everything else not corresponding to the pore size of the resin
beads would not. With the application of Buffer B, it was thought that most of the desired ALDH1A1
would be removed with the first application of buffer B (or the second step), while the second application
(third step) would just clean the column for the next application of the lysate. For the affinity
chromatography, a sufficiently high concentration of imidazole (250 mM) knocked out the ALDH1A1
protein and allowed it to be eluted, as evidenced in our results. At that concentration, the Ni2+
protein lost
interest in the ALDH1A1 and instead took up the imidazole.
For the enzyme kinetics, we needed NAD to be added due to its necessity as a cofactor to
ALDH1A1. The GSH was necessary to keep the cysteine disulfide bonds intact and the pyrazole was
necessary to inhibit the E. coli dehydrogenases. The sodium pyrophosphate buffer and the water provided
a similar cytosol medium in which the ALDH1A1 could interact with the substrate, acetylaldehyde, added
at the last possible minute.
ELISA results for row one were expected to be purple based on BCIP forming a purple
precipitate. Row two showed as anticipated with PNPP resulting in a soluble yellow colored product from
binding with secondary antibody used. TMB gave a blue color as expected from binding with secondary
antibody. Secondary antibody used for ELISA was goat antirabbit AP conjugate.
Western blotting results showed an isolated band at approximately 50kb alongside protein
standard bovine serum albumin (BSA). Minute amount of trailing impurities were detected on the same
lane. This is quite possibly from troubleshooting the gel itself after already loading onto the apparatus due
to the activating strip not taken off prior to run. Other possible contaminants may have been introduced
from faulty aseptic technique.
About the only steps that needed troubleshooting were the ones related to time allotment for
various steps on the last day. Given limited time constraints of the students and instructors, we had to

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shorten some of the steps from a full hour to a half hour in the interest of not staying in the lab until
9:00 pm.
As stated before, we did not expect nearly as good as results as we received due to the earlier
vortexing done to our cells in trying to mix them up following a centrifuge process. However,our results
were better than quite a few of our classmates,possibly due to overloading our size exclusion and nickel
columns with twice as much of the initial setup fluids as recommended. In addition, I also noticed that
many of the classmates had more trouble with the sonicator than we did, so it’s possible that they over or
under sonicated.
It would have been interesting to have tried immunoaffinity chromatography with the ALDH1A1
to run those results in conjunction with the Western blot, dot blot and ELISA, but I’m not sure how the
columns would have been set up or if the costs involved would have been prohibitive.

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Conclusions:
The ALDH1A1 project began with us being completely befuddled and ended with us having a
much greater understanding and recognition of the importance of the ALDH1A1 protein within our
bodies, particularly with the first report and reading all the literature provided by Dr. Rekha. With the
second report, we learned many of the downstream processes involved in purification of a protein for
medical research purposes. In doing so, the ALDH1A1 that we obtained was not quite as pure as we
would have liked, but it appeared to have a higher activity than many of our classmates.
The experiments conducted in the first half, or upstream processing, helped us gain a greater
understanding of what’s involved in a medical research laboratory, especially in reading about the
processes that were implemented prior to our receiving the cells and plasmids. In terms of the techniques
involved, most of what was done in the first half applied loosely to the second, but the equipment and
techniques in the second part provided a much richer lab experience.
If it’s possible, a smaller class size would have worked better with the subject matter and the lab
equipment needed for this project. That being said, we gained a greater understanding of teamwork and
planning out priorities, especially with the exhaustive literature review we undertook in the first half. We
also gained a sense of appreciation for keeping a level temper in the lab, particularly when things didn’t
go as expected or when we messed things up by unnecessary vortexing.
Overall, many of the concepts in this course corresponded closely with the other courses we’re
taking this semester:Cell Culture and Molecular Biology. While spending 12 hours a week in lab was
exhausting in itself, it did provide a solid expectation of what we’ll likely being doing in our next two
years at the U. It also gave us a much better sense of how things can go catastrophically wrong in a lab,
fortunately without the displeasure of actually experiencing it. By the same measure,it also instilled a
sense of accomplishment in that our protein remained active throughout and was verified by five separate
tests.

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of May, 2015 ALDH1A1 Protein Expression Page 12 of 13
Acknowledgements:
We would like to thank Dr. Rekha Ganaganur for her hard work and dedication to the academic
affairsof her classes and the success of her students. We would also like to thank Kelly Theede forher
contributionsto the bettering of this laboratory throughout the semester.
-Jason Morris and Antony Crane

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of May, 2015 ALDH1A1 Protein Expression Page 13 of 13
Sources:
1. Dr. Rehka’s ALDH Project step 5 Laboratory Protocol.
2. Dr. Rekha’s Chromatography Theory Document
3. Dr. Rekha’s ALDH Size Exclusion Chromatography Purification; Getting Nickel Column Ready
Step 6 protocol.
4. Dr. Rekha’s Theory Nickel Chromatography Document.
5. Dr. Rekha’s ALDH Activity Through Enzyme Kinetics Theory Document.
6. Dr. Rekha’s Polyacrylamide Gel Electrophoresis (PAGE) for ALDH Proteins Samples Step 8
protocol.
7. Dr. Rekha’s Polyacrylamide Gel Electrophoresis (PAGE) for ALDH Proteins Samples Spring
2015 Theory Document.
8. Dr. Rekha’s ImmunoAssays with ELISA, Western,and Dot Blot Protocol.
9. Dr. Rekha’s Theory and Study Guide: Immunological Methods Theory Document