Abstract
The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting. In this paper, we report on the production and purification of a soluble anti-HER2 scFv antibody secreted by Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at the 3′-end was inserted into the expression vector pPICZα and transformed in P. pastoris. The highest expression level was obtained in presence of 0.5% methanol and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. pastoris proved very valuable as an expression system, allowing the isolation of 10 mg/L of highly purified antibody, remarkably higher than previously reported data. The functionality of purified anti-HER2 scFv was assessed by cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer cells, showing good affinity and high selectivity for the target membrane receptor. These findings confirm that P. pastoris is a suitable host for high level expression of antibody fragments and highlight the potential role of scFv800E6 in diagnostic and therapeutic application.
Similar content being viewed by others
References
Arbabi-Ghahroudi M, Tanha J, MacKenzie R (2005) Prokaryotic expression of antibodies. Cancer Metastasis Rev 24:501–519
Cai H, Chen L, Wan L, Zeng L, Yang H, Li S, Li Y, Cheng J, Lu X (2009) High-level expression of a functional humanized anti-CTLA4 single-chain variable fragment antibody in Pichia pastoris. Appl Microbiol Biotechnol 82:41–48
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Colombo M, Corsi F, Foschi D, Mazzantini E, Mazzucchelli S, Morasso C, Occhipinti E, Polito L, Prosperi D, Ronchi S, Verderio P (2010) HER2 targeting as a two-sided strategy for breast cancer diagnosis and treatment: outlook and recent implications in nanomedical approaches. Pharmacol Res 62:150–165
Freyre FM, Vázquez JE, Ayala M, Canaán-Haden L, Bell H, Rodríguez I, González A, Cintado A, Gavilondo JV (2000) Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris. J Biotechnol 76:157–163
Galeffi P, Lombardi A, Donato MD, Latini A, Sperandei M, Cantale C, Giacomini P (2005) Expression of single-chain antibodies in transgenic plants. Vaccine 23:1823–1827
Galeffi P, Lombardi A, Pietraforte I, Novelli F, Di Donato M, Sperandei M, Tornambé A, Fraioli R, Martayan A, Natali PG, Benevolo M, Mottolese M, Ylera F, Cantale C, Giacomini P (2006) Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems. J Transl Med 4:39
Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D (2006) Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94:353–361
Inan M, Meagher MM (2001) Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92:585–589
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lannoo N, Vervecken W, Proost P, Rougé P, Van Damme EJ (2007) Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris. Protein Expr Purif 53:275–282
Leath CA 3rd, Douglas JT, Curiel DT, Alvarez RD (2004) Single-chain antibodies: a therapeutic modality for cancer gene therapy (review). Int J Oncol 24:765–771
Lombardi A, Sperandei M, Cantale C, Giacomini P, Galeffi P (2005) Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment. Protein Expr Purif 44:10–15
Mårlind J, Kaspar M, Trachsel E, Sommavilla R, Hindle S, Bacci C, Giovannoni L, Neri D (2008) Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy. Clin Cancer Res 14:6515–6524
Marty C, Scheidegger P, Ballmer-Hofer K, Klemenz R, Schwendener RA (2001) Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris. Protein Expr Purif 21:156–164
Mazzucchelli S, Colombo M, De Palma C, Salvadè A, Verderio P, Coghi MD, Clementi E, Tortora P, Corsi F, Prosperi D (2010) Single-domain protein A-engineered magnetic nanoparticles: toward a universal strategy to site-specific labeling of antibodies for targeted detection of tumor cells. ACS Nano 4:5693–5702
Parker SL, Tong TA, Bolden S, Wingo PA (1996) Cancer statistics, 1996. CA Cancer J Clin 46:5–27
Raemaekers RJ, de Muro L, Gatehouse JA, Fordham-Skelton AP (1999) Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide. Eur J Biochem 265:394–403
Ren F, Li BC, Zhang NN, Cao M, Dan WB, Zhang SQ (2008) Expression, purification and characterization of anti-BAFF antibody secreted from the yeast Pichia pastoris. Biotechnol Lett 30:1075–1080
Rinderknecht M, Villa A, Ballmer-Hofer K, Neri D, Detmar M (2010) Phage-derived fully human monoclonal antibody fragments to human vascular endothelial growth factor-C block its interaction with VEGF receptor-2 and 3. PLoS ONE 5:e11941
Sakai K, Yuasa N, Tsukamoto K, Takasaki-Matsumoto A, Yajima Y, Sato R, Kawakami H, Mizuno M, Takayanagi A, Shimizu N, Nakata M, Fujita-Yamaguchi Y (2010) Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments. J Biochem 147:809–817
Wan L, Cai H, Yang H, Lu Y, Li Y, Li X, Li S, Zhang J, Li Y, Cheng J, Lu X (2008) High-level expression of a functional humanized single-chain variable fragment antibody against CD25 in Pichia pastoris. Appl Microbiol Biotechnol 81:33–41
Yezhelyev MV, Gao X, Xing Y, Al-Hajj A, Nie S, O’Regan RM (2006) Emerging use of nanoparticles in diagnosis and treatment of breast cancer. Lancet Oncol 7:657–667
Zhang G, Liu Y, Hu H (2010) Preparation and cytotoxicity effect of anti-hepatocellular carcinoma scFv immunoliposome on hepatocarcinoma cell in vitro. Eur J Inflamm 8:75–82
Acknowledgments
This work was supported by Regione Lombardia (NanoMeDia Project) and “Fondazione Romeo ed Enrica Invernizzi” and “Centro di Microscopia Elettronica per lo sviluppo delle Nanotecnologie applicate alla medicina” (CMENA).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sommaruga, S., Lombardi, A., Salvadè, A. et al. Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells. Appl Microbiol Biotechnol 91, 613–621 (2011). https://doi.org/10.1007/s00253-011-3306-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-011-3306-3