Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II.
L-Asparaginase (isozyme II) from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. Extracellular expression of recombinant asparaginase was obtained by fusing the gene coding for asparaginase to an efficient pelB leader sequence and an N-terminal 6x histidine tag cloned under the T7lac promoter. Media composition and the induction strategy had a major influence on the specificity and efficiency of secretion of recombinant asparaginase. Induction of the cells with 0.1 mM IPTG at late log phase of growth in TB media resulted in fourfold higher extracellular activity in comparison to growing the cells in LB media followed by induction during the mid log phase. Using an optimized expression strategy a yield of 20,950 UI/L of recombinant asparaginase was obtained from the extracellular medium. The recombinant protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography which gave an overall yield of 95 mg/L of purified protein, with a recovery of 86%. This is approximately 8-fold higher to the previously reported data in literature. The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.
Khushoo
Amardeep
A
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Pal
Yogender
Y
Singh
Bhairab Nath
BN
Mukherjee
K J
KJ
D001215:Asparaginase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D003470:Culture Media; D011994:Recombinant Proteins; D001215:Asparaginase