Data Availability StatementNot applicable. peptide (GBP). The fusion proteins was portrayed in inclusion body form. Subsequently, the denaturation/renaturation procedure and Ni-column purification had been performed. Our data indicated the purified GBP-SubA could bind GRP78 been around on cancers cell surface particularly, internalize into cells to inactivate intracellular GRP78 and induce apoptosis. Furthermore, the apoptosis induction aftereffect of GBP-SubA was improved certainly combined with the elevated cancer tumor cell surface area GBP78. Conclusions It indicates the recombinant GBP-SubA possesses the dual functions of GBP and SubA to induce malignancy cell apoptosis specifically, exposing that GBP-SubA keeps important implications for developing as an anti-cancer peptide drug. Graphical abstract: A schematic representation of the building and function of GBP-SubA.? Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0294-5) contains supplementary material, which is available to authorized users. (STEC) O113:H21 strain 98NK2, and it is a member of Abdominal5 toxins family [16, 17]. The Subtilase cytotoxic holotoxin is composed of one 35 kD catalytic A subunit (SubA) and five 13 kD B subunits (SubB). SubB can bind to glycan receptors (Neu5Gc) that universally exist on mammalian cell surface, and SubB is necessary for internalization of the holotoxin. SubA is the catalytic subunit, its serine protease activity is responsible for toxicity to the sponsor cells [18]. Moreover, SubA possesses the intense substrate specificity. The analysis from proteomics and practical studies reveals that GRP78 is the specific molecular target for SubA. It cleaves GRP78 between the amino acid residues Leu416 and Leu417 that locate within the hinge region between the ATPase and COOH-terminal protein binding domains [19]. The cleavage at this site prospects to loss RO3280 of GRP78 function and exerts fatal effects for the cells [20]. Here, a fusion protein GBP-SubA was constructed and from inclusion body through denaturation and renaturation process. The experiment confirmed the fusion protein kept the native features of GBP and SubA simultaneously. It possessed dual effectiveness of focusing on and killing tumor cells by against GRP78 only, but with less effect on normal cells. This study may provide a fresh strategy for developing targeted anti-tumor medicines. Methods Reagents Plasmid pET-28a was maintained in our laboratory. DNA polymerase, DNA Ligation Kit, and restriction enzymes were from Takara Biotech Co., Ltd. (Dalian, China). The Plasmid Mini Kit and Gel Removal Package were bought from Omega (Norcross, USA). RPMI-1640 moderate and DMEM/F12 (1:1) moderate had been from Hyclone (Logan, USA). Fetal bovine serum (FBS) was from Sangon biotech (Shanghai, China). Antibodies for His-tag and GRP78 had been bought from Proteintech (Wuhan, China). Antibody for GRP78 N-terminal was from Beyotime Biotechnology (Shanghai, China). Phycoerythrin-conjugated RO3280 supplementary antibody was extracted from Santa Cruz Biotechnology (Dallas, USA). Rhodamine Phalloidin was bought from Cytoskeleton (Denver, USA). Guava Nexin Reagent and polyvinylidene difluoride (PVDF) membrane had been from Millipore (Darmstadt, Germany). BCA proteins assay package and Immunol Staining Repair Solution had been from Beyotime (Jiangsu, China). Enhanced chemiluminescence recognition package was from Engreen (Beijing, China). All the chemical substances and reagents had been extracted from Sigma (St. Louis, USA). Cell strains and lines Individual cell lines DLD1, HepG2 and HL-7702 had been extracted from the Cell Loan provider of the Chinese language Academy of Sciences (Shanghai, China). DLD1 and HepG2 cells had been grown up at 37?C in RPMI-1640 moderate supplemented with 10?% high temperature inactivated FBS, 50 IU penicillin and 50 g/mL streptomycin. HL-7702 cells had been grown up at 37?C in DMEM/F12 (1:1) moderate supplemented with 10?% high temperature inactivated FBS, 50 IU penicillin and 50 g/mL streptomycin. The strains DH5, BL21 (DE3) and Rosetta (DE3) had been preserved inside our lab and kept in Luria-Bertani (LB) moderate filled RO3280 with 15?% glycerol at ?80?C. Recombinant plasmid structure The DNA encoding GBP (WIFPWIQL) and SubA (Gene Identification: 3654564) had been fused and synthesized by TaKaRa Biotechnology (Dalian, China), as well as the limitation sites of HI and I had been separately presented to 5 and 3 ends from the fused DNA. The synthesized GBP-SubA DNA portion was ligated into T-Vector pMD19 (TaKaRa, Dalian, China). The recombinant plasmid pMD19-GBP-SubA and plasmid pET-28a had been digested using HI and I in buffer K at 30?C for 2 h. After gel purification and removal, GBP-SubA DNA portion was ligated into family pet-28a vector using DNA Ligation Package with a proportion of put: vector?=?5:1 (mol/mol) CTSL1 because the user manual. Recombinant family pet-28a-GBP-SubA was changed into Rosetta (DE3) cells. Cells were grown in 37 overnight?C on LB plates with kanamycin. Positive colonies had been discovered by colony limitation and PCR digestive function, and confirmed by DNA sequencing (Sangon, Shanghai, China). Appearance from the recombinant proteins Six histidine-tagged fusion proteins GBP-SubA was portrayed in the web host stress Rosetta (DE3) cells. Quickly, Rosetta (DE3) cells filled with family pet-28a-GBP-SubA had been streaked on the LB-agar plate.