Purification of recombinant superoxide dismutase protein from   E. coli cells

 The complete coding sequence of gene was placed in E. coli expression system. The recombinant SOD expressed in prokaryotic system- E. coli, was purified using various steps of protein purification and analysis viz. ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, assay for SOD activity etc. (Figs 1 to 3).  The purified recombinant SOD exhibited single band of ~19 kDa on reducing SDS-PAGE (Fig 1) and showed thermostable property upon assay for SOD activity before and after boiling (Fig 4). 

 

Fig-3: Elution profile of recombinant superoxide dismutase protein on gel filtration column, using high performance liquid chromatography

 Fig-4:  Assay for thermostability of superoxide  dismutase enzyme.

Upscale production  of  recombinant superoxide dismutase protein:

 

Process optimization for over expression of the recombinant SOD at the shake flask level was carried out. Different parameters relevant to E .coli growth and SOD expressions were standardized. Results obtained are compiled in Table-1.

 

Table1:    OPTIMIZED CONDITIONS FOR RECOMBINANT SOD PRODUCTION IN SHAKE FLASK

  

 

PARAMETERS EXAMINED

RESULTS

1.

 

pH

7.0 (Buffered)

2.

Temperature shift

37º  C to 28 º C during Induction

 

3.

Carbon source

Glucose (0.25 % carbon

Equivalent)

4.

Complex Nitrogen Source

Tryptone and Yeast extract

(20g/L + 10g/L)

 

5.

Copper and Zinc conc.

200 ppm and 2 ppm

 

6.

Cell density at time of induction

0.6-0.9

7.

IPTG Conc.

1mM

8.

Total SOD units

50 units

9.

Total Specific Activity

43 Units/total mg of protein

  

Fig-5: Colony PCR of P. pastoris transformants. The amplified bands are positives for SOD gene and correspond to 459 b size insert.