title: [Expression of recombinant human kallistatin in Pichia pastoris by high density cell culture, and its purification and characterization].
abstract: Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.
authors.affiliation: School of Biomedical Science, Institutes of Molecular Medicine, Huaqiao University, Quanzhou 362021, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yong-Mei
authors.initials: YM
authors.lastname: Xing
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Diao
mesh_terms: D019149:Bioreactors; D002196:Capillaries / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D015843:Serpins; C051324:kallistatin