title: Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions.
abstract: Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Gao
authors.forename: Jian
authors.initials: J
authors.lastname: Zhao
authors.forename: Liqiang
authors.initials: L
authors.lastname: Fan
authors.forename: Suxia
authors.initials: S
authors.lastname: Li
authors.forename: Fujun
authors.initials: F
authors.lastname: Wang
authors.forename: Shengli
authors.initials: S
authors.lastname: Ji
authors.forename: Qinsheng
authors.initials: Q
authors.lastname: Yuan
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D019763:Heparin Lyase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019763:Heparin Lyase