title: Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris.
abstract: Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF‑mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Manman
authors.initials: M
authors.lastname: Su
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Weiqin
authors.initials: W
authors.lastname: Chang
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Manhua
authors.initials: M
authors.lastname: Cui
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Shuying
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China.
authors.forename: Tianmin
authors.initials: T
authors.lastname: Xu
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D000432:Methanol / Q000494:pharmacology; D058990:Molecular Targeted Therapy; D009285:Naphthoquinones / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000473:pathology*; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D000970:Antineoplastic Agents; D009285:Naphthoquinones; D009363:Neoplasm Proteins; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; C014638:beta-lapachone; D000432:Methanol