Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies.
The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.
Goyal
Deepika
D
Institute of Microbial Technology, Sector - 39A, Chandigarh 160036, India.
Sahoo
Debendra K
DK
Sahni
Girish
G
D000327:Adsorption; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D002479:Inclusion Bodies / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013300:Streptokinase / Q000302:isolation & purification*
D011994:Recombinant Proteins; D013300:Streptokinase