EXPRESSION, PURIFICATION AND CHARACTERIZATION OF
HUMAN Alpha A CRYSTALLIN
Kayla East
Dr. S. Swamy Mruthinti
Biology Department
Abstract:
Alpha crystallin, found in vertebrate eye lens, is a molecular chaperone. It was
shown to protect other proteins from stress-induced denaturation. The oligomer of α
crystallin is made up of 2 gene products, αA and αB. Mutations on this αA and B are
associated with congenital cataracts. R116C mutation on αA seems to affect is chaperone
function. Aquaporin 0 (AQP0), the water channel exclusively expressed in the eye lens, is
responsible to maintain lens fiber cell homeostasis. The long term goal of this project is
to show the effect of αA mutations on the chaperone function towards AQP0. Immediate
goal is to express human alpha A crystallin using inducible pET-23D(+) expression
system in bacteria. The recombinant wild-type αA crystallin was purified by
chromatography, and characterized by HPLC and immunochemical analysis. Our results
show that we have successfully expressed the wild type αA crystallin. The protein is
predominantly in the cytosolic fraction, which was purified by HPLC. The
immunochemical analysis showed that the expressed αA crystallin eluted as the major
peak with a molecular weight of ~ 800 kda, expected molecular weight of oligomeric
alpha crystallin. Studies are in progress to generate R116C mutants and express the
mutant protein. Both the wild type and mutant protein will be used to study the chaperone
function towards AQP).
The purified protein from this essay will be used in future research to test the
chaperone function of α crystalline with site-directed mutagenesis. Purified protein from
this essay will be used to compare human α crystalline interactions with AQP0 with that
of bovine α crystalline. The purpose of this project is to ultimately find the wild and
mutant type of Alpha A Crystallin. The mutant type R116C is suspected to be the cause
of cataracts. The study of this mutant will help to tell the chaperone function of alpha
crystallin. Cataract formation causes blindness in more than 17 million people
worldwide. Prevention or delaying of cataract development is the main goal of this
research. Alpha crystallin could play a role in maintaining lens transparency because of
its ability to function as a chaperone and having the ability to suppress aggregation which
causes lens opacity.
The cDNA for human αA crystallin was obtained from prior research and used in
these studies. The construction of αA and αB crystallin was done in the following
procedure. αA-crystalline was put into the pGEM-T vector (Promega) and turned into
DH5 α E. Coli cells. The transformed cells were plated on ampicillin agar and incubated
overnight. Α-crystallin DNA was excised from pGEM-T, in positive colonies using Hind
III, Nco I, and Mung Bean and was ligated into the pET23d(+) vector. DH5α E. Coli cells
were transformed with either αA- or αB-pET23d(+) constructs. The DNA was double
digested with Nco I and Hind III and purified with Wizard Mini prep Plus kit (Promega).
The procedure for the Wizard Mini prep kit is as follow: 5 ml of the DNA solution is
distributed into four test tubes then put into the centrifuge for five minutes at 10000
rounds per minute. The supernatant (liquid left after centrifugation) is then poured off and
disposed of. 250 micro liters of cell lysis sln is then added and re-suspended by vortexing
or pipetting. 250 micro liters of cell lysis sln is also added. The product is then incubated
for 1-5 minutes. 10 micro liters of alkaline protease sln is added and then incubated at
room temperature for 5 minutes. 350 micro liters of neutralization sln is added and the
bacteria lysate is put into the centrifuge at max speed for 10 minutes at room temperature.
DNA sequencing was done with the T7 promoter primer in a 3700 ABI automated
DNA sequencing (Medical College of Georgia- Core Facility). Sterile Falcon 2059
(Promega) transformation culture tubes were chilled on ice. Frozen competent BL21 p.lys
cells were put on Ice for 5 min, and 50 μL was quickly pipetted into the Falcon 2059
tubes. 2 μL DNA was added to each tube, and the tubes were left on ice for 10 min. The
tubes were “heat shocked” for 45 sec at exactly 42°C. The tubes were returned to ice for
2 minutes. 450 μL SOC medium (promega) was added to each tube, and the tubes were
incubated for 1 hour at 37°C with shaking. Transformed cells were then plated on lb agar
plates containing both Ampicillin and Chloramphenicol, and were incubated over night.
The expression plasmids (αA-pET23d(+) and αB-pET23d(+) ) vectors were transformed
into E. coli BL21 cells. The cells were grown in cultures until the reacted an optimum
growth at an OD600 of 0.6.
The cells were Induced with two amounts of IPTG and grown for differentiating
time increments. Both sets were grown in increments of 4Hr, 6Hr, and overnight with one
set having been induced with 20 μL IPTG and the other set induced with 40 μL IPTG. 10
ml Luria Broth (starter culture w/ 50 microgram/ml ampicillin[Fisher] and 34
microgram/ml chloramphenicol [sigma]) was inoculated with 30 micro liters glycerol
stock containing desired plasmid. Then it was grown overnight at 37 degrees in a shaking
incubator. The product was used to inoculate 500 ml of Luria Broth with appropriate
antibiotics. It was then incubated at 37 degrees with shaking until the OD equals 0.6. T7
promoter is induced using 0.5 mM isopropyl-1-thio-B-D-galactopyranoside Promega
(IPTG). The cells were then harvested and re-suspended in 10 ml of cold lysis buffer. The
cells were then sonicated three times each to burst open the cells. Cells were then left at
room temperature for one hour and then put into the centrifuge at 28000 rpm for 45
minutes. Supernatant was then filtered through a .2 micro meter filter and used for
purification of expressed crystallin.
The results for these procedures are done by a couple of processes through protein
assay kits, running gels and staining. The Protein Assay Kit was ran approximately three
times. Protein Assay Kit procedure was done by adding 40 micro liters of solution B, 2
ml of solution A, 10 micro liter of either supernatant 1, supernatant 2,pellet, cells or all of
the above, and 10 micro liters of PBS solution. For the blank in the procedure, 40 micro
liters of B is added, 2 ml of A and 100 micro liters of PBS solution. After this kit was
run, the solutions had to be incubated for 30 minutes at 37 degrees Celsius. The solutions
were then put onto a spectrometer to obtain the optical density which helps to calculate
the percent of proteins in a solution.
Amounts of each solution were then calculated to see how much was needed to
be put into the SDS page gels. The SDS page gels were ran on July 3, 5 and 12. On July
3, the order of the gel was from well 1-10: marker, cell, supernatant 1, pellet, 4th hour
high, 4th hour low, 6th hour high, 6th hour low, overnight high and overnight low. The gel
on July 5 was ran in wells 1-10 with different amounts in each well. The order was: 10
micro liters of marker, 5 micro liters of supernatant, 5 micro liters of pellet, 10 micro
liters of supernatant, 10 micro liters of pellet, blank, 5 micro liters of supernatant, 5 micro
liters of pellet, 10 micro liters of supernatant, and 10 micro liters of the pellet. July 12 gel
procedure was as follows: 10 ml of marker, 5 micro liters of pellet, 5 micro liters of
supernatant, 20 micro liters of peak A III, 5 micro liters of Peak A II, blank, 5 micro liters
of pellet, 5 micro liters of pellet, 20 micro liters of Peak A III and 5 micro liters of Peak
A II. With each of the gels run, the first five wells in the gel were then processed using a
staining solution and put into a shaking incubator overnight to bring out the lines of the
wells.
After the supernatant was analyzed on SDS-PAGE gels it was probed with both
αA- and αB-antibodies to show the presence of α-crystallin. Alpha crystallin was
expressed used three different actions: western blot, dot blot and the anti-body procedure.
The last four lines of the wells were then put into another machine that was ran for a few
hours. The outcome of this was the beginning of the western blot procedure. The dot blot
procedure was as follows: samples of 5 micro liters were spotted on the nitrocellulose
strip and allowed to air dry. The papers of the western blot and of the dot blot were then
put into dry low fat milk and were left for a couple of hours or overnight. The liquids are
then poured off and the first antibody (15ml Antibody Dialution Buffer and 3 micro liters
alpha a) is then added to the blots. The antibody is left in the shaking incubator for
around one hour and then pour antibody back into container. Wash with TBST for 5
minutes 3 times in the incubator and disregard liquid each time. Add the second antibody
(15 ml antibody dialution buffer and 3 micro liters goat anti-rabbit antibody and leave in
incubator for approximately one hour. Remove antibody two and place back in container
again and repeat washes with TBST. Then AP Buffer is put into the solution for 5 min to
wash completely before reagent is added. Substrate Phosphate Reagent Kit (10 ml AP
Buffer, 100 micro liter solution A, and 100 micro liters solution B) is added to the
container and left in the incubator for 5 to 10 minutes and then shake manually until
reagent is complete and the color forms on papers.
Expressed cells were cultured in 500mL of agar containing both ampicillin and
Chloramphenicol. The contents were left to grow until they reached an O.D. of .6 and
then were distributed into 4 tubes and centrifuged for 10 minutes at 15000 rpm, and then
combined into one centrifuge tube using 10 mL of cell lysis buffer. The cells were then
subjected to two freeze thaw cycles, followed by two sonication cycles for 30 sec at 80
cycles per minute. The cells were then re-centrifuged for 15 minutes at 15000rpm. The
supernatant and pellet were then collected in separate tubes and run on SDS page gels.
A 600 x 7.8 mm BIOSEP-SEC3000 column (Phenomenex) was used with Waters
625 LC System. The Supernatant solution was processed through the HPLC machine
along with HPLC Buffer, to separate out the particles by molecular size. There was a
flow rate of 1 ml/min and each minute’s sample was collected into a different test tubes.
As the solution was processed through the machine, the results were simultaneously
graphed on a chromatogram. This process was repeated three times to total 3 ml for each
minute. The peaks from the graph were analyzed and the test tubes containing the
solution from each peak were combined for a more concentrated solution. The solutions
were then run using the dot blot procedure. This graph is from the HPLC machine that
shows that different proteins in the solutions.
In the project, we have successfully been implemented out the gene into the pET23d(+) vector. Also, the alpha crystallin has been expressed using IPTG, as seen
through the SDS page gels and immunochemical analysis. The protein has also been
characterized successfully and has also been purified through HPLC machine. This
project was already started and the main goal of this project was to continue on the
project. The purified protein from this essay will be used in future research to test the
chaperone function of α crystalline with site-directed mutagenesis.
Purified protein from this essay will be used to compare human α crystalline
interactions with AQP0 with that of bovine α crystalline.
REFERENCES:
Shroff, Nilufer P. Molecular Chaperone Function of Site-Directed Mutants of
AlphaA and AlphaB-Crystallins. Unpublished procedure. June 1999.