INTRODUCTION:

The term 'molecular chaperonin' was carried out to describe the specealized function of the nuclear protein nucleoplasmin in promoting chromatin assembly (Laskey et al.,1978). Chaperonion control and catalysed post translational folding, assembley, or membrane translocation of some protein (Hartl et al.,1994). The most throughly studied chaperonin system is the GroEL and GroES from E.coli (Tilly and Georgopoulos,1982) studied invivo and invitro (Ellis,1987).

Chaperonin belongs to an ubiquitous class of protein that shows a high degree of sequence identity (Hartl and Martin,1995). Chaperonin GroEL and GroES from E.coli being a ubiquitous class of sequence related molecules in bacteria (Mehra et al.,1986) and organelles chloroplast(Hemmingsen et al.,1988), mitochondria(Pickettos et al.,1989) cytoplasm of eukaryotes(Hendrick and Hartl,1993) and archebacteria(Macarion and Mcrio,1994; Gupta,1995).

Most studies on chaperonin has been carried out with chaperonin from mesophilic organism such as E.coli chaperonin with high thermostability naturally exit in thermophiles. Purification of chaperonin from thermophilic bacterium Thermus aquaticus (Mukulik and Benda,1993), Thermo anaerobacter brocku (Truscott et al.,1994) from E.coli(Llorca et al.,1994) has been studied.

In over expressing strain E.coli. U1/pUS01/pUSS1∆CAT, GroES consist of a single heptameric ring of 10 kD and GroEL have two stack ring of both seven identical subunit of 60 kD surrounding a cavity of 90 kD globular protein(Oscar Llorca et al.,1996). The GroES and GroEL protein act in a concerted way in a ATP dependent manner because GroEL has ATPase activity that prevents their non-specefic aggregation during spontaneous folding to their native conformation. They increase the rate of folding product but not increases the rate of protein folding (Hartl and Martin,1995).

MATERIALS AND METHODS:

Chaperonin GroES and GroEL was purified to near homogeneity following method of Buchner et al.(1991) from E.coli U1/pus01/puss1∆ CAT(Buchner et al.,1991). Fractionation and purification was achieved by using collumn of Q-sepharose FF and sephacry S-200 HR. Purification was monitered by 12.5% SDS-PAGE(Laemmli, 1970) visualized by coomassie BB R-250(Merril,1990) and silver staining (Sambrooke et al.,2001) and further confirmed by western blotting(Burnette,1981). Protein was estimated by Bradford(1976) method.

Purification:

GroES and GroEL was purified to near homogeneity as following Cell Growth: Over producing strain E.coli U1/pus01/puss1∆ CAT, generation time was found to 78 mins were grown in LB (Luria-Broth) to an O.D. ₅₇₈of about 1.0. Media contains antibiotics ampicillin 100µl/ml, kanamycin 20µl/ml and streptomycin 30µl/ml.

IPTG induction:

The GroESL gene was induced by the addition of 2mM IPTG added 2 hrs before cell harvesting. Cell were harvested at 7000rpm using GSA and SS-34 roters and store it at -20⁰C till further use.

Table 2. GroES details at different Steps

Steps	GroES (mg)	Absolute(%)
Crude extract after sonication	1360	100
Crude extract after heat denaturation	605	44.7
After Q-Sepharose FF	85	6.29
After Q-Sephacryl S-200 HR	50 mg	3.7

~50 mgs GroES purified from 1.4 litres culture of E.coli. U1/pUS01/pUSS1 ∆ CAT.

Table 3. GroEL details at different steps

Steps	GroEL (mg)	Absolute(%)
Crude extract after sonication	9555	100
Crude extract after heat denaturation	5200	54
After Q-Sepharose FF	12.7	0.13
After Q-Sephacryl S-200 HR	7.5 mg	0.078

7.5 mgs GroEL recovery after purification from 1.4 litres culture of E.coli. U1/pUS01/pUSS1 ∆CAT.

Sonication:

Cells were suspended in ice cold buffer-A (5mg/5ml). It was stir for 30 mins at room temp. Carefully to avoid foam forming. Cell were lysed by sonication using ½” disrupter horn submerged 1-1/2” in the solution. The cell tube is kept in the ice bath during sonication. The general sonication period is 10-20 mins.

Sonication setting:

Cycle time : 10-20 mins Duty cycle : 60 Out put : 6-7

The cell lisate is centrifuge for 20 mins at 15000 rpm at 4⁰C. The cell pellet is further resuspended in buffer-A, sonicated and centrifuged again as above.

Heat treatment:

Crude exract was then heated to 65⁰C for 30 mins to remove most of the heat labile coagulated proteins, centrifuged it to remove coagulated proteins. It was analysed by 12.5% SDS-PAGE to check the expression and purity.

GroES purification:

IPTG induced cells, after cell lysis, heat treatment and centrifugation was loaded to Q-sepharose FF column, equilibriated with buffer-A, the conductivity and pH of starting buffer and column eluent was found same. When it was eluted with the 0-1 M NaCl, GroES aws eluted at 0.2

M NaCl. Fraction purity were checked by 12.5% SDS-PAGE, minor contaminats were removed by size exclusive Sephacryl S-200 HR further purity and fraction were checked by 12.5% SDS-PAGE(Table 2, Figs.1and2, Graph 1).

GroEL purification:

IPTG induced cells sonicated, heat treatment and centrifugation extract were Amicon concentrated and loaded to Sephacryl S-200 HR and GroEL rich fraction were pooled and further purified by Q-sepharose FF. To separate GroEL from bound proteins the protein solution was treated with 10mM ATP for 20 mins at 60⁰C. Final purification was achieved by using sephacryl S-200 HR. Purity were analysed by 12.5% SDS-PAGE, a clear distinct band of 60 kD compare from maker(Table 3, Fig 3) .

Western blotting: (Burnette,1981) GroEL:

The purified GroEL was resolved on 12.5% mini SDS-PAGE and was semi dried, blotted to nitrocellulose paper, and after washing, primary antibodies were incubated, again unbound antibodies were washed. Secondary antibodies anti-IgG from rabbit were applied after 3-4 washing, incubated with substrate solution and finally develop, it was stopped by rinsing TDW. The blot was dried and store within a light protected sheet(Fig 4).

Solution: buffer A:

50 mM Tris_HCl pH7.5, 2mM EDTA, 2mM MgCl_2,0.1 mg/ml DNase ;

Washing solution:

IX TBS+20pH 7.4; Substrate solution: Nitrobenzene tetrazolium=4.5 µl, 5-bromo 4-chloro 3-indolyl Phosphate= 3.5µl in 1x TBS pH 9.4; Incubation solution: IX TBS-Twin 20 pH 9.4; Antibody: anti rabbit IgG alkaline phosphatase conjugate developed in goat.

RESULTS:

Purified GroES and GroEL analysed in 12.5% SDS-PAGE was found in close proximity to the marker proteins Lysozyme 14.3 kD and BSA 66 kD respectively confirm their molecular weight of ~10 kD and ~60kD. The purification was achieved using anion exchange Q-Sephrose FF and size exclusive Sephacryl S-200 HR chromatography (Buchner et al., 1991).

DISCUSSION:

The genetically modified E. coli. U1/pUS01/ pUSS1∆CAT contain two plasmids pUS 01 codes for resistant to ampicillin is pACYC 177 based plasmid. While pUSS1 code for resistant to kanamycin it is apBR322 based plasmid. The E coli. U1/pUS01/ pUSS1∆CAT was constructed where groESL operon was replaced by that of B. sterothermophile. This strain is perfectally viable, demonstrating that the B. sterothermophile operon is functionally interchagable with that of E.coli. For efficient coding of GroES and GroEL protein it’s GroESL operon was fused to an IPTG inducible promoter.

ACKNOWLEDGEMENT:

I am very thankful to Prof GP Paul MBU, IMS,BHU for providing GM bacterial strain, chemicals and lab facilities. My sincere thanks to Smt. Annapurna Chatterjee for her essential technical and moral support.

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Protein	ε 276M^-1 cm^-1	Protein	ε 276M^-1 cm^-1	Protein	ε 276M^-1 cm^-1
GroELEC	11500	GroEL Bt	12300	GroES	11200