Dept. of Biology, UWG, Carrollton, GA

EXPRESSION, PURIFICATION AND CHARACTERIZATION OF HUMAN A CRYSTALLIN

Students: Kayla East & Jessica Phillips

Faculty Advisor: Dr. S. Swamy Mruthinti, Department of Biology

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 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 ran using the dot blot procedure.

Conclusions

1)We have successfully implemented out the gene into the pET-23d(+) vector.

2) We have successfully expressed alpha crystalline using IPTG, as seen through the SDS page gels and immunochemical analysis.

3) We have successfully characterized our protein.

4) 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 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.

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 wildtype 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).

Acknowledgments: a special thanks to the National Science Foundation STEP grant #DUE-0336571.

Experimental ProcedurecDNA for Human alpha A crystallin: The cDNA for human αA crystallin was obtained from prior research and used in these studies.

Construction of αA and αB Crystallins: αA-crystalline was ligated into the pGEM-T vector (Promega) and transformed into DH5 α E. Coli cells. 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)

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 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.

Expression of αA and αB Crystallins: 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 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 α-crystallin: After expression, the cells were centrifuged at 5000 RPM for 5 minutes. The cells were then lysed using a probe to release the α-crystalline into the supernatant. The cellular debris was settled again and the supernatant was analyzed on SDS-PAGE gels and probed with both αA- and αB-antibodies to show the presence of α-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.

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.

Supernatant I

Peak A III

Uninduced IPTG induced

Expression of alpha crystallin: αcrystallin 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.

Supernatant 1

Peak A

Immunoreactivity against αA Crystallin was seen in the supernatant and Peak A of HPLC. Note that none of the other peaks from HPLC showed any immunoreactivity.

Peak B

Peak C

Peak D

Peak F

Peak E

Peak APeak B

Peak D

Peak E

Peak F

Pellet

Buffer

Peak C

α A