Expression of VGRNb-PE immunotoxin in transplastomic lettuce (Lactuca sativa L.)
- 220 Downloads
This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation.
Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7 µg per 100 mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.
KeywordsAngiogenesis Immunotoxin Chloroplast transformation Lettuce
The authors gratefully thank Dr. Babak Latif for creation of pBL102 vector, Dr. Mostafa Modarresi for assistance in creating pBL VGRNb-PE, Reza Moazzami for assistance with protein purification, and Ehsan Ali-Rahimi for help with the cell culture experiments.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Behdani M, Zeinali S, Karimpour M, Khanahmad H, Schoonooghe S, Aslemarz A, Seyed N, Moazami-Godarzi R, Baniahmad F, Habibi-Anbohi M, Hassanzadeh-Ghassabeh G, Muyldermans S (2013) Development of VEGFR2-specific nanobody Pseudomonas exotoxin A conjugated to provide efficient inhibition of tumor cell growth. New Biotechnol 30:205–209. https://doi.org/10.1016/j.nbt.2012.09.002 CrossRefGoogle Scholar
- Chebolu S, Daniell H (2007) Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis. Plant Biotechnol J 5:230–239. https://doi.org/10.1111/j.1467-7652.2006.00234.x CrossRefPubMedPubMedCentralGoogle Scholar
- Daniell H, Ruiz G, Denes B, Sandberg L, Langridge W (2009) Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function. BMC Biotechnol 9:33. https://doi.org/10.1186/1472-6750-9-33 CrossRefPubMedPubMedCentralGoogle Scholar
- Davoodi-Semiromi A, Schreiber M, Nalapalli S, Verma D, Singh ND, Banks RK, Chakrabarti D, Daniell H (2010) Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol J 8:223–242. https://doi.org/10.1111/j.1467-7652.2009.00479.x CrossRefPubMedGoogle Scholar
- Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J 1:71–79. https://doi.org/10.1046/j.1467-7652.2003.00008.x CrossRefPubMedGoogle Scholar
- Gisby MF, Mellors P, Madesis P, Ellin M, Laverty H, O’Kane S, Ferguson MWJ, Day A (2011) A synthetic gene increases TGFb3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant Biotechnol J 9:618–628CrossRefPubMedGoogle Scholar
- Harada H, Maoka T, Osawa A, Hattan JI, Kanamoto H, Shindo K, Otomatsu T, Misawa N (2014) Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic Res 2:11–23. https://doi.org/10.1007/s11248-013-9750-3 Google Scholar
- Kazemi-Lomedasht F, Behdani M, Bagheri KP, Habibi-Anbouhi M, Abolhassani M, Arezumand R, Shahbazzadeh D, Mirzahoseini H (2015) Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody. Mol Immunol 65:58–67. https://doi.org/10.1016/j.molimm.2015.01.010 CrossRefPubMedGoogle Scholar
- Lim S, Ashida H, Watanabe R, Inai K, Kim YS, Mukougawa K, Fukuda H, Tomizawa K, Ushiyama K, Asaon H, Tamoi M, Masutani H, Shigeoka S, Yodoi J, Yokota A (2011) Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts. Plant Mol Biol 76:335–344. https://doi.org/10.1007/s11103-011-9745-5 CrossRefPubMedGoogle Scholar
- Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
- Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H (2007) Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts—oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J 5:495–510. https://doi.org/10.1111/j.1467-7652.2007.00259.x CrossRefPubMedPubMedCentralGoogle Scholar
- Sambrook J, Russell DR (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338. https://doi.org/10.1038/73796 CrossRefPubMedGoogle Scholar
- Verma D, Moghimi B, LoDuca PA, Singh HD, Hoffman BE, Herzog RW, Daniell H (2010) Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice. Proc Natl Acad Sci USA 107:7101–7106. https://doi.org/10.1073/pnas.0912181107 CrossRefPubMedPubMedCentralGoogle Scholar