ALDH1A1_V02_JasonMorris_14May2015

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

ABSTRACTThis 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, 2015In partial fulfillment of Biochemistry Laboratory and Techniques Class Project under the supervision of Dr. Rekha Ganaganur, spring semester, 2015

11th of May, 2015 ALDH1A1 Protein Expression Page 1 of 14

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.


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.


Various technical procedures conducted with the following instruments:

- Fisher Scientific Sonic Dismembrator; Model 500; Serial #BCKO 6082J04; Room S3600; Lot # BIOT-0165

-BECKMAN COULTER Allegra Centrifuge; Model 64R; Serial # ALV08D06; ID # BIOT-CN03

-Mini-PROTEAN TGX Polyacrylamide Gel by BIORAD; Exp 2015-12-11; Catalog # 456-1094

-VWR Orbital Shaker; ID # BIOT-0101; Serial # 41694

-CPS Controller; ID # BIOT-0128; Serial # A1039430483TK; Room S3300

- Bio-Rad Labs SmartSpec Plus Spectrophotometer; Serial # 273 BR 07396

- Beckman Coulter Allegra X-22 R Centrifuge; Serial # ALB03F019; ID # BIOT-0118

-Eppendorf Centrifuge; Model 5415D; Serial # 5425-26984; ID # BIOT-0115

-Isotemp Fisher Scientific; Model 2052FS Dryblock heater; Serial # 1649080344320


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)

Pictured: Sonicator with sample being sonicated.

Pictured: Size Exclusion column


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


reagent


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)

Our results: Top gray line:

size exclusion sample

Top blue line:

Buffer C3 sample

Pictured: Buffer C3 and Buffer B


SDS-PAGE gel:

1 2 3 4 5 6 7 8 9 10

Lane 1: marker loaded with 15 uL

Lane 2: Uninduced IPTG with 38 uL

Lane 3: Induced IPTG with 38 uL

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.


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 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 IPTG with 38 uL

Lane 3: Induced IPTG with 38 uL

Lane 4: Before size exclusion with 38 uL

Lane 5: Post size exclusion (buffer B) with 38 uL




Lane 9: D buffer with 38 uL

Lane 10: BSA protein standard with 38 uL

Pictured: Western Blot


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 several student groups for substrate BCIP.

Pictured: Dot Blot

Pictured: 1st and 3rd rows of ELISA

Pictured: 1st and 2nd rows of ELISA


Discussions:

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


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.


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.


Acknowledgements:

We would like to thank Dr. Rekha Ganaganur for her hard work and dedication to the academic

affairs of her classes and the success of her students. We would also like to thank Kelly Theede for her

contributions to the bettering of this laboratory throughout the semester.

-Jason Morris and Antony Crane


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

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ALDH1A1_V02_JasonMorris_14May2015