Preliminary research on the expression, purification and function of the apoptotic fusion protein, Sival

Asian Pac J Cancer Prev. 2014;15(20):8685-8. doi: 10.7314/apjcp.2014.15.20.8685.

Abstract

The objective of the present study was to investigate cloning, expression, and functions of the recombinant protein, Siva1. Siva1 gene was synthesized by RT-PCR from HCT116 cells. Plasmids were cleaved with the restriction endonuclease, BamH1/Sal1 and products were connected to pQE30, which underwent cleavage by BamH1/Sal1. The recombinant plasmid, pQE30-Siva1, was identified after digestion with restriction endonucleases followed by transformation into E. coli M15. Expression of Siva1 was induced by IPTG and identified by SDS- PAGE following purification with affinity chromatography. The results showed that size of Siva1 was 12 kDa, consistent with the molecular weight of the His-Siva1 fusion protein. Functional test demonstrated that Siva1 significantly inhibited the invasion and migration of HCT116 cells. It may thus find clinical application for control of cancers.

MeSH terms

  • Apoptosis Regulatory Proteins / genetics*
  • Apoptosis Regulatory Proteins / isolation & purification*
  • Cell Movement / genetics
  • Cloning, Molecular / methods*
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / physiopathology
  • Electrophoresis, Polyacrylamide Gel / methods
  • Escherichia coli / genetics
  • Gene Expression Regulation, Neoplastic*
  • Genetic Engineering / methods
  • HCT116 Cells / cytology
  • HCT116 Cells / physiology
  • Humans
  • Plasmids / genetics
  • Real-Time Polymerase Chain Reaction / methods
  • Recombinant Fusion Proteins / genetics
  • Sensitivity and Specificity
  • Tumor Cells, Cultured

Substances

  • Apoptosis Regulatory Proteins
  • Recombinant Fusion Proteins
  • SIVA1 protein, human