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Fusion to elastin-like polypeptide increases production of bioactive human IFN-γ in tobacco

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Abstract

The plant-based expression systems are now accredited as bioreactors for the high production of various biopharmaceuticals. However, low levels of agglomeration and the absence of effective procedures for purification of recombinant proteins have remained two essential obstacles in molecular farming. In this research, we have studied the production of human interferon gamma (hIFN-γ) in tobacco and analyzed the effects of elastin-like polypeptide (ELP) tag and subcellular localization on its accumulation. We report a remarkable enhancement of accumulation of the fusion proteins versus the corresponding unfused hIFN-γ proteins. Furthermore, the hIFN-γ (with and without ELP) accumulated to higher levels in the endoplasmic reticulum. The ELP fusion proteins were successfully recovered from total soluble protein with adding 2.75 M NaCl and three rounds of inverse transition cycling (ITC). The hIFN-γ was also separated from ELP with Enterokinase cleavage of the fusion protein and recovered by ITC. Inverse transition analysis indicated that the hIFN-γ-ELP variants aggregate above their inverse transition temperature and at high ionic strength. Investigation of glycosylation revealed that fused or unfused hIFN-γ proteins are N-glycosylated in different cellular locations. Moreover, N-glycosylation analysis and bioassay showed that fusion to ELP does not disturb glycosylation process and antiviral activity of hIFN-γ.

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Abbreviations

BSA:

Bovine serum albumin

CaMV:

Cauliflower mosaic virus

CHO:

Chinese hamster ovary

CPMV:

Cowpea mosaic virus

DAB:

3, 3′-Diaminobenzidine

ELISA:

Enzyme-linked immunosorbent assay

ELP:

Elastin-like polypeptides

ER:

Endoplasmic reticulum

Ext:

Extensin signal peptide

hIFN-γ:

Human interferon-γ

ITC:

Inverse transition cycling

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

nos :

Nopaline synthase

PTM:

Post-translation modification

PVP:

Polyvinyl pyrrolidone

qRT-PCR:

Quantitative real-time PCR

TSP:

Total soluble protein

Tt:

Inverse transition temperature

UTR:

Untranslated regions

VSV:

Vesicular stomatitis virus

Zera:

γ-Zein signal peptide

References

  • Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H (2007) Field production and functional evaluation of chloroplast-derived interferon-α2b. Plant Biotechnol J 5:511–525

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bortesi L, Rademacher T, Schiermeyer A, Schuster F, Pezzotti M, Schillberg S (2012) Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells. BMC Biotechnol 12:40

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    CAS  Google Scholar 

  • Chen TL, Lin YL, Lee YL, Yang NS, Chan MT (2004) Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures. Transgenic Res 13:499–510

    CAS  PubMed  Google Scholar 

  • Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE (2009a) Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants. Biotechnol Bioeng 103:562–573

    CAS  PubMed  Google Scholar 

  • Conley AJ, Mohib K, Jevnikar AM, Brandle JE (2009b) Plant recombinant erythropoietin attenuates inflammatory kidney cell injury. Plant Biotechnol J 7:183–199

    CAS  PubMed  Google Scholar 

  • Dirisala VR, Nair RR, Srirama K, Reddy PN, Rao KRSS, Kumar NSS, Parvatam G (2017) Recombinant pharmaceutical protein production in plants: unraveling the therapeutic potential of molecular pharming. Acta Physiol Plant 39:18

    Google Scholar 

  • Ebrahimi N, Memari HR, Ebrahimi MA, Ardakani MR (2012) Cloning, transformation and expression of human gamma interferon gene in tomato (Lycopersicon esculentum Mill.). Biotechnol Biotechnol Equip 26:2925–2929

    CAS  Google Scholar 

  • Egelkrout E, Rajan V, Howard JA (2012) Overproduction of recombinant proteins in plants. Plant Sci 184:83–101

    CAS  PubMed  Google Scholar 

  • Egito AS, Girardet JM, Miclo L, Gaillard JL (2001) Highly sensitive periodic acid/Schiff detection of bovine milk glycoproteins electrotransferred after nondenaturing electrophoresis, urea electrophoresis, and isoelectric focusing. Le Lait 81:775–785

    CAS  Google Scholar 

  • Floss DM, Sack M, Stadlmann J, Rademacher T, Scheller J, Stöger E, Fischer R, Conrad U (2008) Biochemical and functional characterization of anti-HIV antibody–ELP fusion proteins from transgenic plants. Plant Biotechnol J 6:379–391

    CAS  PubMed  Google Scholar 

  • Floss DM, Sack M, Arcalis E, Stadlmann J, Quendler H, Rademacher T, Stoger E, Scheller J, Fischer R, Conrad U (2009) Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody. Plant Biotechnol J 7:899–913

    CAS  PubMed  Google Scholar 

  • Gao Y, Yang J, Cai Y, Fu S, Zhang N, Fu X, Li L (2018) IFN-γ-mediated inhibition of lung cancer correlates with PD-L1 expression and is regulated by PI3K-AKT signaling. Int J Cancer. https://doi.org/10.1002/ijc.31357

    Article  PubMed  PubMed Central  Google Scholar 

  • Heidari-Japelaghi R, Haddad R, Garoosi GA (2011) Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol Biotechnol 49:129–137

    Google Scholar 

  • Heidari-Japelaghi R, Haddad R, Valizadeh M, Dorani-Uliaie E, Jalali-Javaran M (2019a) Elastin-like polypeptide fusions enhance transient expression of human IFN-γ in tobacco leaves. S Afr J Bot 125:321–328

    CAS  Google Scholar 

  • Heidari-Japelaghi R, Haddad R, Valizadeh M, Dorani-Uliaie E, Jalali-Javaran M (2019b) Elastin-like polypeptide fusions for high-level expression and purification of human IFN-γ in Escherichia coli. Anal Biochem 585:113401

    CAS  PubMed  Google Scholar 

  • Heidari-Japelaghi R, Valizadeh M, Haddad R, Dorani-Uliaie E, Jalali-Javaran M (2020) Production of bioactive human IFN-γ protein by agroinfiltration in tobacco. Protein Expr Purif 173:105616

    CAS  PubMed  Google Scholar 

  • Höglund M (1998) Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)—what is the difference? Med Oncol 15:229–233

    PubMed  Google Scholar 

  • Joensuu JJ, Conley AJ, Lienemann M, Brandle JE, Linder MB, Menassa R (2009) Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana. Plant Physiol 152:622–633

    PubMed  Google Scholar 

  • Kaldis A, Ahmad A, Reid A, McGarvey B, Brandle J, Ma S, Jevnikar A, Kohalmi SE, Rima Menassa R (2013) High-level production of human interleukin-10 fusions in tobacco cell suspension cultures. Plant Biotechnol J 11:535–545

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ke C, Xiong H, Zhao C, Zhang Z, Zhao X, Rensing C, Zhang G, Yang S (2019) Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-019-09638-w

    Article  PubMed  Google Scholar 

  • Knight M, Braverman J, Asfaha K, Gronert K, Stanley S (2018) Lipid droplet formation in Mycobacterium tuberculosis infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense. PLoS Pathog 14:e1006874

    PubMed  PubMed Central  Google Scholar 

  • Kontsek P, Martens E, Vandenbroeck K, Kontseková E, Waschütza G, Sareneva T, Billiau A (1997) Structural immuno-analysis of human and porcine interferon gamma: identification of shared antigenic domain. Cytokine 9:550–555

    CAS  PubMed  Google Scholar 

  • Leister P, Tileva M, Krachmarova E, Nacheva G (2014) Expression of human interferon-gamma gene in human tissue culture cells. Biotechnol Biotechnol Equip 27:3573–3576

    Google Scholar 

  • Lowther W, Lorick K, Lawrence SD, Yeow WS (2012) Expression of biologically active human interferon alpha 2 in Aloe vera. Transgenic Res 21(6):1349–1357

    CAS  PubMed  Google Scholar 

  • Luchakivskaya Y, Kishchenko O, Gerasymenko I, Olevinskaya Z, Simonenko Y, Spivak M, Kuchuk M (2011) High-level expression of human interferon alpha-2b in transgenic carrot (Daucus carota L.) plants. Plant Cell Rep 30:407–415

    CAS  PubMed  Google Scholar 

  • Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17:1112–1115

    CAS  PubMed  Google Scholar 

  • Mignone F, Gissi C, Liuni S, Pesole G (2002) Untranslated regions of mRNAs. Genome Biol 3:1–10

    Google Scholar 

  • Mori M, Zhang GH, Kaido M, Okuno T, Furusawa I (1993) Efficient production of human gamma interferon in tobacco protoplasts by genetically engineered brome mosaic virus RNAs. J Gen Virol 74:1255–1260

    CAS  PubMed  Google Scholar 

  • Murakami A, Ohnishi K (2012) Target molecules of food phytochemicals: food science bound for the next dimension. Food Funct 3:462–476

    CAS  PubMed  Google Scholar 

  • Nakiboneka R, Mugaba S, Auma BO, Kintu C, Lindan C, Nanteza MB, Kaleebu P, Serwanga J (2019) Interferon gamma (IFN-γ) negative CD4+ and CD8+ T-cells can produce immune mediators in response to viral antigens. Vaccine 37:113–122

    CAS  PubMed  PubMed Central  Google Scholar 

  • Phan HT, Pohl J, Floss DM, Rabenstein F, Veits J, Le BT, Chu HH, Hause G, Mettenleiter T, Conrad U (2013) ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice. Plant Biotechnol J 11:582–593

    CAS  PubMed  Google Scholar 

  • Raucher D, Chilkoti A (2001) Enhanced uptake of a thermally responsive polypeptide by tumor cells in response to its hyperthermia-mediated phase transition. Cancer Res 61:7163–7170

    CAS  PubMed  Google Scholar 

  • Reuter LJ, Bailey MJ, Joensuu JJ, Ritala A (2014) Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. Plant Biotechnol J 12:402–410

    CAS  PubMed  Google Scholar 

  • Sainsbury F, Lomonossoff GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148:1212–1218

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sareneva T, Mortz E, Tolo H, Roepstorff P, Julkunen I (1996) Biosynthesis and N-glycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition. Eur J Biochem 242:191–200

    CAS  PubMed  Google Scholar 

  • Sarker A, Rathore AS, Gupta RD (2019) Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process. Microb Cell Fact 18:5

    PubMed  PubMed Central  Google Scholar 

  • Scheller J, Henggeler D, Viviani A, Conrad U (2004) Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Transgenic Res 13:51–57

    CAS  PubMed  Google Scholar 

  • Scheller J, Leps M, Conrad U (2006) Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol J 4:243–249

    CAS  PubMed  Google Scholar 

  • Sousa DA, Mulder KCL, Nobre KS, Parachin NS, Franco OL (2016) Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag. J Biotechnol 234:83–89

    CAS  PubMed  Google Scholar 

  • Trabbic-Carlson K, Liu L, Kim B, Chilkoti A (2004) Expression and purification of recombinant proteins from Escherichia coli: comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. Protein Sci 13:3274–3284

    CAS  PubMed  PubMed Central  Google Scholar 

  • Urry DW (1988) Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics. J Protein Chem 7:1–34

    CAS  PubMed  Google Scholar 

  • Urry DW (1997) Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J Phys Chem B 101:11007–11028

    CAS  Google Scholar 

  • Urry DW, Parker TM, Reid MC, Gowda DC (1991) Biocompatibility of the bioelastic materials, poly(GVGVP) and its gamma-irradiation cross-linked matrix—summary of generic biological test results. J Bioact Compat Polym 6:263–282

    CAS  Google Scholar 

  • Wang K (2006) Methods in molecular biology, Agrobacterium protocols. In: Clemente T (ed) Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana), vol 1, 2nd edn. Humana Press, Totowa, NJ, pp 143–154

    Google Scholar 

  • Wu Y, Zhao D, Song L, Xu W (2009) Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants. Biologia 64:1115–1122

    CAS  Google Scholar 

  • Zhang X, Urry DW, Daniell H (1996) Expression of an environmentally friendly synthetic protein-based polymer gene in transgenic tobacco plants. Plant Cell Rep 16:174–179

    PubMed  Google Scholar 

  • Zhou Y, Li X, Yan D, Peprah FA, Ji X, Fletcher EE et al (2019) Multifunctional elastin-like polypeptide renders β-glucosidase enzyme phase transition and high stability. Biotechnol Biofuels 12:157

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was financially supported by Grant No: 951105 of the Biotechnology Development Council of the Islamic Republic of Iran.

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Authors and Affiliations

  1. Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

    Reza Heidari-Japelaghi, Mostafa Valizadeh & Ebrahim Dorani-Uliaie

  2. Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran

    Raheem Haddad

  3. Department of Plant Breeding, Faculty of Agriculture, University of Tarbiat Modares, Tehran, Iran

    Mokhtar Jalali-Javaran

Authors
  1. Reza Heidari-Japelaghi
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  2. Mostafa Valizadeh
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  4. Ebrahim Dorani-Uliaie
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  5. Mokhtar Jalali-Javaran
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Correspondence to Reza Heidari-Japelaghi.

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Heidari-Japelaghi, R., Valizadeh, M., Haddad, R. et al. Fusion to elastin-like polypeptide increases production of bioactive human IFN-γ in tobacco. Transgenic Res 29, 381–394 (2020). https://doi.org/10.1007/s11248-020-00205-y

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