title: Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells.
abstract: BACKGROUND
  Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells.
  
  
  METHODS
  MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-β-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments.
  
  
  RESULTS
  Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner.
  
  
  CONCLUSIONS
  It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.
authors.affiliation: Department of Urology, Xuzhou Central Hospital, Xuzhou, Jiangsu Province, China - zpying58@126.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Hlin
authors.forename: Zhang
authors.initials: Z
authors.lastname: Zhi-Guo
authors.forename: Han
authors.initials: H
authors.lastname: Cong-Hui
authors.forename: Zhao
authors.initials: Z
authors.lastname: Yan
authors.forename: Liang
authors.initials: L
authors.lastname: Qing
authors.forename: Jiang
authors.initials: J
authors.lastname: Bo
authors.forename: He
authors.initials: H
authors.lastname: Hou-Guang
authors.forename: Zhang
authors.initials: Z
authors.lastname: Jun-Jie
authors.forename: Zhang
authors.initials: Z
authors.lastname: Pei-Ying
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D029777:Momordica charantia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D054790:Ribosome Inactivating Proteins, Type 2 / Q000096:biosynthesis* / Q000627:therapeutic use*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy*
chemical_list: C066347:MAP30 protein, Momordica charantia; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2