EXPRESSION, PURIFICATION AND CHARACTERIZATION OF HUMAN aA CRYSTALLIN

1
EXPRESSION, PURIFICATION AND CHARACTERIZATION OF
HUMAN aA CRYSTALLIN Students Kayla East
Jessica Phillips Faculty Advisor Dr. S. Swamy
Mruthinti, Department of Biology
Uninduced
IPTG induced
Expression of alpha crystallin acrystallin was
cloned into the pET23d expression vector down
stream the T7 promoter. The promoter was induced
with 0.5 mM IPTG. Cells were collected by
centrifugation and analyzed on 12 SDS-PAGE gels.
Lane 1 uninduced and the lane 2 is IPTG induced.
Expression of alpha crystallin was seen on only
the IPTG induced cells, showed by arrow as the 22
KD protein.
Buffer
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 a
crystallin is made up of 2 gene products, aA and
aB. Mutations on this aA and B are associated
with congenital cataracts. R116C mutation on aA
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 aA
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 aA
crystallin was purified by chromatography, and
characterized by HPLC and immunochemical
analysis. Our results show that we have
successfully expressed the wildtype aA
crystallin. The protein is predominantly in the
cytosolic fraction, which was purified by HPLC.
The immunochemical analysis showed that the
expressed aA 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).
Pellet
Supernatant I
Peak A III
Peak A II
Dot Blot Samples (5 ul) were spotted on the
nitrocellulose strip and allowed to air dry. The
strip was incubated with polyclonal antibody
raised against alpha crysallin and the reactivity
was visualized by adding the the substrate
mixture for alkaline phosphatase.
a A
Experimental Procedure cDNA for Human alpha A
crystallin The cDNA for human aA crystallin was
obtained from prior research and used in these
studies. Construction of aA and aB Crystallins
aA-crystalline was ligated into the pGEM-T vector
(Promega) and transformed into DH5 a E. Coli
cells. Transformed cells were plated on
ampicillin agar and incubated overnight.
?-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.
DH5a E. Coli cells were transformed with either
aA- or aB-pET23d() constructs. The DNA was
double digested with Nco I and Hind III and
purified with Wizard Mini prep Plus kit
(Promega) DNA sequencing DNA sequencing was
done with the T7 promoter primer in a 3700
ABI automated DNA sequencing (Medical College of
Georgia- Core Facility). Transformation of
BL21 Cells 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 42C. 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 37C with shaking. Transformed cells were
then plated on lb agar plates containing both
Ampicillin and Chloramphenicol, and were
incubated over night. Expression of aA and aB
Crystallins The expression plasmids
(aA-pET23d() and aB-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
then Induced with two amounts of IPTG and grown
for differentiating time increments. Both sets
were grown in increments of 1Hr, 3Hr, and
overnight with one set having been induced with
20 µL IPTG and the other set induced with 40 µL
IPTG. Isolation of a-crystallin After
expression, the cells were centrifuged at 5000
RPM for 5 minutes. The cells were then lysed
using a probe to release the a- crystalline into
the supernatant. The cellular debris was settled
again and the supernatant was analyzed on
SDS-PAGE gels and probed with both aA- and
aB-antibodies to show the presence of
a-crystallin. Protocol for protein
purification 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 cycl es 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.
Supernatant 1
Peak A
Peak B
Peak C
Peak D
Peak E
Peak E
Peak F
Immunoreactivity against aA Crystallin was seen
in the supernatant and Peak A of HPLC. Note that
none of the other peaks from HPLC showed any
immunoreactivity.
Peak D
Peak F

• Conclusions
• We have successfully implemented out the gene
  into the pET-23d() vector.
• We have successfully expressed alpha crystalline
  using IPTG, as seen through the SDS page gels and
  immunochemical analysis.
• We have successfully characterized our protein.
• We have successfully purified our protein.
• Future Goals
• The purified protein from this essay will be used
  in future research to test the chaperone function
  of a crystalline with site-directed mutagenesis.
• Purified protein from this essay will be used to
  compare human a crystalline interactions with
  AQP0 with that of bovine a crystalline.

Peak C
Peak A
Peak B
HPLC Purification 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
minutes 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 ran using the
dot blot procedure.
Acknowledgments a special thanks to the National
Science Foundation STEP grant DUE-0336571.