Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris

Su,Manman; Chang,Weiqin; Zhang,Kun; Cui,Manhua; Wu,Shuying; Xu,Tianmin

doi:10.3892/or.2015.4448

Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris

Authors:
- Manman Su
- Weiqin Chang
- Kun Zhang
- Manhua Cui
- Shuying Wu
- Tianmin Xu
View Affiliations

Affiliations: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China
Published online on: November 25, 2015 https://doi.org/10.3892/or.2015.4448
Pages: 1179-1185

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Raghuraman H and Chattopadhyay A: Melittin: A membrane-active peptide with diverse functions. Biosci Rep. 27:189–223. 2007. View Article : Google Scholar
2	Sommer A, Fries A, Cornelsen I, Speck N, Koch-Nolte F, Gimpl G, Andrä J, Bhakdi S and Reiss K: Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation. J Biol Chem. 287:23678–23689. 2012. View Article : Google Scholar : PubMed/NCBI
3	Son DJ, Lee JW, Lee YH, Song HS, Lee CK and Hong JT: Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacol Ther. 115:246–270. 2007. View Article : Google Scholar : PubMed/NCBI
4	Wang C, Chen T, Zhang N, Yang M, Li B, Lü X, Cao X and Ling C: Melittin, a major component of bee venom, sensitizes human hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating CaMKII-TAK1-JNK/p38 and inhibiting IkappaBalpha kinase-NFkappaB. J Biol Chem. 284:3804–3813. 2009. View Article : Google Scholar
5	Jo M, Park MH, Kollipara PS, An BJ, Song HS, Han SB, Kim JH, Song MJ and Hong JT: Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway. Toxicol Appl Pharmacol. 258:72–81. 2012. View Article : Google Scholar
6	Liu S, Yu M, He Y, Xiao L, Wang F, Song C, Sun S, Ling C and Xu Z: Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology. 47:1964–1973. 2008. View Article : Google Scholar : PubMed/NCBI
7	Russell PJ, Hewish D, Carter T, Sterling-Levis K, Ow K, Hattarki M, Doughty L, Guthrie R, Shapira D, Molloy PL, et al: Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: In vitro and in vivo studies. Cancer Immunol Immunother. 53:411–421. 2004. View Article : Google Scholar : PubMed/NCBI
8	Soman NR, Baldwin SL, Hu G, Marsh JN, Lanza GM, Heuser JE, Arbeit JM, Wickline SA and Schlesinger PH: Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth. J Clin Invest. 119:2830–2842. 2009. View Article : Google Scholar : PubMed/NCBI
9	Moghadam BY, Hou WC, Corredor C, Westerhoff P and Posner JD: Role of nanoparticle surface functionality in the disruption of model cell membranes. Langmuir. 28:16318–16326. 2012. View Article : Google Scholar : PubMed/NCBI
10	Funayama JC, Pucca MB, Roncolato EC, Bertolini TB, Campos LB and Barbosa JE: Production of human antibody fragments binding to melittin and phospholipase A2 in Africanised bee venom: Minimising venom toxicity. Basic Clin Pharmacol Toxicol. 110:290–297. 2012. View Article : Google Scholar
11	Holle L, Song W, Holle E, Wei Y, Li J, Wagner TE and Yu X: In vitro- and in vivo-targeted tumor lysis by an MMP2 cleavable melittin-LAP fusion protein. Int J Oncol. 35:829–835. 2009.PubMed/NCBI
12	Botkjaer KA, Deryugina EI, Dupont DM, Gårdsvoll H, Bekes EM, Thuesen CK, Chen Z, Ploug M, Quigley JP and Andreasen PA: Targeting tumor cell invasion and dissemination in vivo by an aptamer that inhibits urokinase-type plasminogen activator through a novel multifunctional mechanism. Mol Cancer Res. 10:1532–1543. 2012. View Article : Google Scholar : PubMed/NCBI
13	Li J, Lin Y, Zhuang H and Hua ZC: Expression, purification, and biological characterization of the amino-terminal fragment of urokinase in Pichia pastoris. J Microbiol Biotechnol. 23:1197–1205. 2013. View Article : Google Scholar : PubMed/NCBI
14	Yang L, Sajja HK, Cao Z, Qian W, Bender L, Marcus AI, Lipowska M, Wood WC and Wang YA: uPAR-targeted optical imaging contrasts as theranostic agents for tumor margin detection. Theranostics. 4:106–118. 2013. View Article : Google Scholar
15	Ding Y, Zhang H, Zhong M, Zhou Z, Zhuang Z, Yin H, Wang X and Zhu Z: Clinical significance of the uPA system in gastric cancer with peritoneal metastasis. Eur J Med Res. 18:282013. View Article : Google Scholar : PubMed/NCBI
16	Thummarati P, Wijitburaphat S, Prasopthum A, Menakongka A, Sripa B, Tohtong R and Suthiphongchai T: High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis. World J Gastroenterol. 18:244–250. 2012. View Article : Google Scholar : PubMed/NCBI
17	Foekens JA, Peters HA, Look MP, Portengen H, Schmitt M, Kramer MD, Brünner N, Jänicke F, Meijer-van Gelder ME, Henzen-Logmans SC, et al: The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res. 60:636–643. 2000.PubMed/NCBI
18	Kim TD, Song KS, Li G, Choi H, Park HD, Lim K, Hwang BD and Yoon WH: Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer. BMC Cancer. 6:2112006. View Article : Google Scholar : PubMed/NCBI
19	Laufs S, Schumacher J and Allgayer H: Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease. Cell Cycle. 5:1760–1771. 2006. View Article : Google Scholar : PubMed/NCBI
20	Rockway TW, Nienaber V and Giranda VL: Inhibitors of the protease domain of urokinase-type plasminogen activator. Curr Pharm Des. 8:2541–2558. 2002. View Article : Google Scholar : PubMed/NCBI
21	Carriero MV and Stoppelli MP: The urokinase-type plasminogen activator and the generation of inhibitors of urokinase activity and signaling. Curr Pharm Des. 17:1944–1961. 2011. View Article : Google Scholar : PubMed/NCBI
22	Li H, Soria C, Griscelli F, Opolon P, Soria J, Yeh P, Legrand C, Vannier JP, Belin D, Perricaudet M, et al: Amino-terminal fragment of urokinase inhibits tumor cell invasion in vitro and in vivo: Respective contribution of the urokinase plasminogen activator receptor-dependent or-independent pathway. Hum Gene Ther. 16:1157–1167. 2005. View Article : Google Scholar : PubMed/NCBI
23	Longuespée R, Boyon C, Desmons A, Vinatier D, Leblanc E, Farré I, Wisztorski M, Ly K, D'Anjou F, Day R, et al: Ovarian cancer molecular pathology. Cancer Metastasis Rev. 31:713–732. 2012. View Article : Google Scholar : PubMed/NCBI
24	Su D, Katsaros D, Xu S, Xu H, Gao Y, Biglia N, Feng J, Ying L, Zhang P, Benedetto C, et al: ADP-ribosylation factor-like 4C (ARL4C), a novel ovarian cancer metastasis suppressor, identified by integrated genomics. Am J Transl Res. 7:242–256. 2015.PubMed/NCBI
25	Coosemans A, Baert T and Vergote I: A view on dendritic cell immunotherapy in ovarian cancer: How far have we come? Facts Views Vis Obgyn. 7:73–78. 2015.PubMed/NCBI
26	Sun D, Sun M, Zhu W, Wang Z, Li Y and Ma J: The anti-cancer potency and mechanism of a novel tumor-activated fused toxin, DLM. Toxins. 7:423–438. 2015. View Article : Google Scholar : PubMed/NCBI