Design, purification and assessment of GRP78 binding peptide-linked Subunit A of Subtilase cytotoxic for targeting cancer cells

BMC Biotechnol. 2016 Sep 1;16(1):65. doi: 10.1186/s12896-016-0294-5.

Abstract

Background: Targeted therapies for cancer, especially the malignant cancer, are always restricted by the deficiency of tumor-specific drug delivery methods. Subtilase cytotoxic is a virulent cytotoxin, and the subunit A (SubA) of it is able to destroy the structure of glucose-regulated protein 78 (GRP78) to induce cell apoptosis, and to be expected as anti-cancer drugs, however, the ubiquitous receptor of subunit B of Subtilase cytotoxic (SubB) restricts its application on cancer therapy.

Results: The present study constructed and expressed a fusion protein of GBP-SubA in E. coli Rosetta (DE3) system, in which the subunit B of Subtilase cytotoxic was replaced by GRP78 binding peptide (GBP). The fusion protein was expressed in inclusion body form. Subsequently, the denaturation/renaturation process and Ni-column purification were performed. Our data indicated the purified GBP-SubA could bind GRP78 existed on cancer cell surface specifically, internalize into cells to inactivate intracellular GRP78 and induce apoptosis. Moreover, the apoptosis induction effect of GBP-SubA was enhanced obviously along with the increased cancer cell surface GBP78.

Conclusions: It indicates that the recombinant GBP-SubA possesses the dual functions of GBP and SubA to induce cancer cell apoptosis specifically, revealing that GBP-SubA holds important implications for developing as an anti-cancer peptide drug. A schematic representation of the construction and function of GBP-SubA.

Keywords: Apoptosis; Cancer; GRP78 binding peptide; Glucose-regulated protein 78; Subunit A of Subtilase cytotoxic.

MeSH terms

  • Antineoplastic Combined Chemotherapy Protocols / pharmacology*
  • Apoptosis / drug effects*
  • Cell Survival / drug effects
  • Cytotoxins / chemistry
  • Cytotoxins / isolation & purification
  • Cytotoxins / pharmacology*
  • Dose-Response Relationship, Drug
  • Drug Design*
  • Escherichia coli Proteins / administration & dosage*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / pharmacokinetics
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / pharmacology
  • Hep G2 Cells
  • Humans
  • Neoplasms, Experimental / drug therapy*
  • Neoplasms, Experimental / metabolism
  • Neoplasms, Experimental / pathology
  • Protein Engineering
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / pharmacology
  • Subtilisins / administration & dosage*
  • Subtilisins / genetics
  • Subtilisins / pharmacokinetics
  • Treatment Outcome

Substances

  • Cytotoxins
  • Escherichia coli Proteins
  • Heat-Shock Proteins
  • Recombinant Fusion Proteins
  • Subtilisins
  • subtilase cytotoxin, E coli
  • molecular chaperone GRP78