title: A novel method for high-level production of psychrophilic TAB5 alkaline phosphatase.
abstract: Heat labile alkaline phosphatases (APs) are widely used in biomedical research for they can easily be heat inactivated once they have done their job. Here we reported a novel method for high-level production of recombinant psychrophilic Antarctic bacterium strain TAB5 alkaline phosphatase (TAP) in Escherichia coli. We synthesized the whole TAP gene according to the synonymous codon choice that is optimal for the E. coli translational system. Then the gene was cloned into pT7 expression vector, expressed in BL21 (DE3) pLysS strain by auto-induction system. The recombinant protein was purified by Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 90.9 mg protein from 16.2 g wet cells in 1L culture medium with the purity over 99%, 340 times as many mg/L (and 21 times the mg/g cells) compared to previous methods. The dephosphorylation activity assay showed that the purified recombinant TAP has similar activity to calf intestinal alkaline phosphatase in room temperature, and it can be totally inactivated by treatment at 60°C for 15 min. Our research provides a novel method for high-level expression, purification and characterization of TAP which is sufficient for high throughput genome analysis and may replace the widely used shrimp AP because of its low cost.
authors.affiliation: Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Zhisheng
authors.initials: Z
authors.lastname: Lu
authors.forename: Wentao
authors.initials: W
authors.lastname: Chen
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Hu
authors.forename: Yu
authors.initials: Y
authors.lastname: Ding
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics*; D000864:Antarctic Regions; D001419:Bacteria / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D000469:Alkaline Phosphatase