title: Expression and purification of L-asparaginase from Escherichia coli and the inhibitory effects of cyclic dipeptides.
abstract: L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from Echerichia coli and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene ansB encoding L-asparaginase was amplified from the genome of E. coli BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10-4mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10-5mol/L.
authors.affiliation: a College of Life Sciences , Qingdao University , Qingdao , P.R. China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: a College of Life Sciences , Qingdao University , Qingdao , P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.affiliation: a College of Life Sciences , Qingdao University , Qingdao , P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D001215:Asparaginase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004151:Dipeptides; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D001215:Asparaginase
keywords: Echerichia coli; L-asparaginase; cyclic dipeptides; expression; purification