Expression, Purification and Characterization of Recombinant Histidine-tagged L-asparaginase II

	Expression, Purification and Characterization of Recombinant Histidine-tagged L-asparaginase II
Article 9, Volume 56, Issue 3, September 2016, Page 707-722 PDF (374.54 K)
Document Type: Regular issue (Original Article)
DOI: 10.21608/ejbo.2016.2728
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Abstract
ESCHERICHIA coli has two L-asparaginase isozymes that have ..... been designated L-asparaginase I and L-asparaginase II. The amino acid sequences of both are rather dissimilar except for a few regions of significant homology. The sequence corresponding to the mature (ansB) was subcloned into pQE-30 expression vector and expressed in E. coli M15. The recombinant histidine-tagged (ansB) was purified to homogeneity by Ni–NTA affinity chromatography and displayed a single 36.0 kDa band on SDS-PAGE. Results revealed that the recombinant His-tagged L-ASNase II was expressed in an active form and its specific activity was estimated to be 286 U/mg. The optimum temperature was attained at 40°C. The enzyme was maximum at pH 7-8. Also the enzyme was stable at 40-50°C. The purified functional enzyme exhibited a specific activity at 286 U/mg and inhibited the growth of human myeloid leukemia cell line (HL-60) with IC50 value of 0.14±0.03 U/ml.
Keywords
Anti-tumor; Cytotoxicity; expression; His-tag; L-asparaginase; Leukemia; Purification; Recombinant


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