Skip to main content

Advertisement

SpringerLink
Log in

Tobacco Chloroplasts as Bioreactors for the Production of Recombinant Superoxide Dismutase in Plants, an Industrially Useful Enzyme

  • Original Paper
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

An inexpensive source of industrially useful enzymes is critical for their commercial production. We have produced an industrially valuable recombinant superoxide dismutase (SOD) in tobacco chloroplasts. A gene from Withania somnifera, encoding a highly stable Cu/Zn SOD, was cloned into a chloroplast transformation vector. It expressed the SOD in tobacco chloroplasts following transformation. The transplastomic plants accumulated the recombinant SOD at up to ∼9 % of the total soluble protein in leaves. The purified chloroplast-expressed recombinant SOD had an estimated specific activity of ∼4600 U/mg. Like the native enzyme, purified recombinant enzyme, prepared from tobacco leaves, was highly stable at high temperatures and tolerated a wide pH range, SDS, ethanol and protease treatment. The results establish the potential of chloroplast transformation for commercial production of recombinant SOD in plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bafana A, Dutt S, Kumar S, Ahuja PS (2010) Superoxide dismutase: an industrial perspective. Crit Rev Biotechnol 31:65–76

    Article  PubMed  Google Scholar 

  • Bally J, Paget E, Droux M, Job C, Dubald M (2008) Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins. Plant Biotechnol J 6:46–61

    CAS  PubMed  Google Scholar 

  • Barnes D, Franklin S, Schultz J, Henry R, Brown E, Coragliotti A, Mayfield SP (2005) Contribution of 5′ and 3′ untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol Gen Genomics 274:625–636

    Article  CAS  Google Scholar 

  • Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

    Article  CAS  PubMed  Google Scholar 

  • Bock R (2013) Strategies for metabolic pathway engineering with multiple transgenes. Plat Mol Biol 83:21–31

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Chen C-N, Pan S-M (1996) Assay of superoxide dismutase activity by combining electrophoresis and densitometry. Bot Bull Acad Sin 37:107–111

    CAS  Google Scholar 

  • Daniell H (2006) Production of biopharmaceuticals and vaccines in plants via the chloroplast genome. Biotechnol J 1:1071–1079

    Article  CAS  PubMed  Google Scholar 

  • Daniell H, Cohill P, Kumar S, Dufourmantel N (2004) Chloroplast genetic engineering. In: Daniell H, Chase C (eds) In molecular biology and biotechnology of plant organelles. Springer, Verlag, pp 443–490

    Chapter  Google Scholar 

  • Dufourmantel N, Tissot G, Garc¸on F, Pelissier B, Dubald M (2006) Stability of soybean recombinant plastome over six generations. Transgenic Res 15:305–311

    Article  CAS  PubMed  Google Scholar 

  • Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97–112

    Article  CAS  PubMed  Google Scholar 

  • Hefferon K (2013) Plant-derived pharmaceuticals for the developing world. Biotechnol J 8:1193–1202

    CAS  PubMed  Google Scholar 

  • Hennig A, Bong K, Roitsch T, Warzecha H (2007) Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis. FEBS J 274:5749–5758

    Article  CAS  PubMed  Google Scholar 

  • Hood EE (2002) From green plants to industrial enzymes. Enz Microb Technol 30:279–283

    Article  CAS  Google Scholar 

  • Khanna P, Staba J (1968) Antimicrobials from plant tissue cultures. Llyodia 31:180–189

    Google Scholar 

  • Kolotilin I, Kaldis A, Pereira EO, Laberge S, Menassa R (2013) Optimization of transplastomic production of hemicellulases in tobacco: effects of expression cassette configuration and tobacco cultivar used as production platform on recombinant protein yields. Biotechnol Biofuels 6:65

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect Immun 73:8266–8274

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kusnadi A, Nikolov G, Howard J (1997) Production of recombinant proteins in plants: practical considerations. Biotechnol Bioeng 56:473–484

    Article  CAS  PubMed  Google Scholar 

  • Madanala R, Gupta V, Deeba F, Upadhyay SK, Pandey V, Singh PK, Tuli R (2011) A highly stable Cu/Zn superoxide dismutase from Withania somnifera plant: cloning, expression and characterization of recombinant protein. Biotechnol Let 33:2057–2063

    Article  CAS  Google Scholar 

  • Madanala R, Gupta V, Singh PK, Tuli R (2012) Development of chloroplast transformation vectors, and a new target region in the tobacco plastid genome. Plant Biotechnol Rep 6:77–87

    Article  Google Scholar 

  • Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nugent JM, Joyce SM (2005) Producing human therapeutic proteins in plastids. Curr Pharm Des 11:2459–2470

    Article  CAS  PubMed  Google Scholar 

  • Peter OO, Shaukat HR (1989) A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lac2 gene in Escherichia coli. J Biolog Chem 264:16973–16976

    Google Scholar 

  • Prashanth SR, Sadhasivam V, Parida A (2008) Over expression of cytosolic copper/zinc superoxide dismutase from a mangrove plant Avicennia marina in Indica rice var Pusa Basmati-1 confers abiotic stress tolerance. Transgenic Res 17:281–291

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Har:old Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sawant SV, Singh PK, Tuli R (2000) Pretreatment of micropojectiles to improve the delivery of DNA in plant transformation. Biotechniques 29:246–248

    CAS  PubMed  Google Scholar 

  • Scharff LB, Bock R (2014) Synthetic biology in plastids. Plant J 78:783–798

    Article  CAS  PubMed  Google Scholar 

  • Streatfeld SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15

    Article  Google Scholar 

  • Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix JD, Ajam T, Surzycki S (2009) Factors effecting expression of vaccines in microalgae. Biologicals 37(3):133–138

    Article  CAS  PubMed  Google Scholar 

  • Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci U S A 90:913–917

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tiwari S, Verma PC, Singh PK, Tuli R (2009) Plants as bioreactors for the production of vaccine antigens. Biotechnol Adv 27(4):449–67

  • Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, vanWijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res 31:1174–1179

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tuli R, Sangwan RS, Kumar S, Bhattacharya S, Misra L, Mandal C, Raghubir R, Nath C, Trivedi PK, Tiwari SK, Mishra P, Chaturvedi P, Sangwan NS, Nair KN, Rawat AKS, Srivatsava V, Srivatsava RK, Ojha SK, Mehrotra S, Khanuja A, Suri KA (2009) A monograph, Aswagandha (Withania somnifera) a model Indian medicinal plant. (eds.) Tuli, R., Sangwan, R.S., CSIR, New Delhi, India. September, 2009. ISBN no: 978-93-80235

  • Verma D, Kanagaraj A, Jin S, Singh ND, Kolattukudy PE, Daniell H (2010) Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars. Plant Biotechnol J 8:332–350

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yarbakht M, Jalali-Javaran M, Nikkhah M, Mohebodini M (2014) Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast. Biotechnol Appl Biochem. doi:10.1002/bab.1230

    PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank the Task Force (CMM004) Programme of CSIR, India, for promoting research on ‘utilization of animals and plants as bioreactors’ and NBRI, Lucknow, India, for providing research facility. RT thanks the Department of Science & Technology for JC Bose Fellowship. VG is thankful to CSIR for senior research fellowship. Authors are thankful to Basant Kumar Dubey and Rajesh Srivastava for the technical support.

Author information

Authors and Affiliations

  1. National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR), Rana Pratap Marg, Lucknow, 226001, UP, India

    Raju Madanala, Ashutosh Kumar Pandey, Subhi Srivastava, Vivek Pandey & Pradhyumna Kumar Singh

  2. Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research, Kukrail Picnic Road, Lucknow, 226015, UP, India

    Vijayta Gupta

  3. Department of Biotechnology, National Agri-Food Biotechnology Institute, C-127, Phase VIII, Industrial Area, SAS Nagar, Mohali, 160071, Punjab, India

    Rakesh Tuli

Authors
  1. Raju Madanala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vijayta Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashutosh Kumar Pandey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Subhi Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vivek Pandey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pradhyumna Kumar Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rakesh Tuli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raju Madanala.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 404 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Madanala, R., Gupta, V., Pandey, A.K. et al. Tobacco Chloroplasts as Bioreactors for the Production of Recombinant Superoxide Dismutase in Plants, an Industrially Useful Enzyme. Plant Mol Biol Rep 33, 1107–1115 (2015). https://doi.org/10.1007/s11105-014-0805-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11105-014-0805-2

Keywords

Search

Navigation