Skip to main content
SpringerLink
Log in

Wolffia arrhiza as a promising producer of recombinant hirudin

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza, an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium-mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia, the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9 ng g−1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.

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

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable for that section.

References

  • Bagdy D, Barabas E, Graf L, Petersen TE, Magnusson S (1976) Hirudin. Methods Enzymol 45:669–678

    CAS  PubMed  Google Scholar 

  • Bertran K, Moresco K, Swayne DE (2015) Impact of vaccination on infection with Vietnam H5N1 high pathogenicity avian influenza virus in hens and the eggs they lay. Vaccine 33:1324–1330

    CAS  PubMed  Google Scholar 

  • Boehm R, Kruse C, Veste D, Barth S, Schnabl H (2001) A transient transformation system for duckweed Wolffia columbiana using Agrobacterium-mediated gene transfer. J Appl Bot 75:107–111

    Google Scholar 

  • Boothe JG, Parmenter DL, Saponja JA (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Dev Res 42:172–181

    CAS  Google Scholar 

  • Boyhan D, Daniell H (2011) Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol J 9(5):585–598

    CAS  PubMed  Google Scholar 

  • Burkhardt PK, Beyer P, Wünn J, Klöti A, Armstrong GA, Schledz M, Lintig J, Potrykus I (1997) Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin a biosynthesis. Plant J 11(5):1071–1078

    CAS  PubMed  Google Scholar 

  • Cabanos C, Ekyo A, Amari Y, Kato N, Kuroda M, Nagaoka S, Takaiwa F, Utsumi S, Maruyama N (2013) High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier. Transgenic Res 22(3):621–629

    CAS  PubMed  Google Scholar 

  • Chang JY (1991) Stability of hirudin, a thrombin-specific inhibitor. The structure of alkaline-inactivated hirudin. J Biol Chem 266(17):10839–10843

    CAS  PubMed  Google Scholar 

  • Chang WC, Chiu PL (1976) Induction of callus from fronds of duckweed (Lemna gibba L.). Bot Bull Acad Sinica 17:106–109

    Google Scholar 

  • Chang WC, Chiu PL (1978) Regeneration of Lemna gibba G3 through callus culture. Zeitschrift Schriftfür Pflanzen Ph 89:91–94

    CAS  Google Scholar 

  • Chaudhary S, Parmenter DL, Moloney MM (1998) Transgenic Brassica carinata as a vehicle for the production of recombinant proteins in seeds. Plant Cell Rep 17(3):195–200

    CAS  PubMed  Google Scholar 

  • Chen HY, Qi XH, Geng X, Xu OG, Wang J, Wu ZR (2012) Expression, purification and characterization of the recombinant hirudin variant III in the Bacillus subtilis. Adv Mater Res 343–344:753–763

    Google Scholar 

  • Cox K, Sterling J, Regan J, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597

    CAS  PubMed  Google Scholar 

  • Daniell H, Streatfieldb SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci J 6(5):219–226

    CAS  Google Scholar 

  • Delaney D, Jilka J, Barker D, Irwin P, Poage M, Woodard S, Horn M, Vinas A, Beifuss K, Barker M, Wiggins B, Drees C, Harkey R, Nikolov Z, Hood E, Howard J (2003) Production of aprotinin in transgenic maize seeds for the pharmaceutical and cell culture markets. In: Vasil IK (ed) Plant Biotechnology 2002 and Beyond. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-2679-5_80

    Chapter  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1(4):19–21

    CAS  Google Scholar 

  • Dickey LF, Gasdaska JR, Cox KM, Peele CG, Spencer D (2009) Expression of monoclonal antibodies in duckweed. United States Patent US7632983B2

  • Dickey LF, Gasdaska JR, Cox KM (2011) Expression of biologically active polypeptides in duckweed. United States Patent US20060195946A1

  • Dodt J, Schmitz T, Schafer T, Bergmann C (1986) Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. FEBS Lett 202(2):373–377

    CAS  PubMed  Google Scholar 

  • Dolgov S, Mikhaylov R, Serova T, Shulga O, Firsov A (2010) Pathogen-derived methods for improving resistance of transgenic plums (Prunus domestica L.) for Plum pox virus infection. Julius-Kühn- Archiv 427:133–140

    Google Scholar 

  • Dolgov SV, Chernobrovkina MA, Khvatkov PA (2013) Composition of medium for culturing duckweed plants (Wolffia arrhiza) in vitro. Russian Patent RU2472338C1

  • Firsov A, Tarasenko I, Mitiouchkina T, Ismailova N, Shaloiko L, Vainstein A, Dolgov S (2015) High-yeld expression of M2e peptide of avian influenza virus H5N1 in transgenic duckweed plants. Mol Biotechnol 57:653–661

    CAS  PubMed  Google Scholar 

  • Firsov A, Tarasenko I, Mitiouchkina T, Shaloiko L, Kozlov O, Vinokurov L, Rasskazova E, Murashev A, Vainstein A, Dolgov S (2018) Expression and immunogenicity of M2e peptide of avian influenza virus H5N1 fused to ricin toxin b chain produced in duckweed plants. Front Chem 6:22

    PubMed  PubMed Central  Google Scholar 

  • Gasdaska JR, Spenser D, Dickey L (2003) Advantages of therapeutic protein production in the aquatic plant Lemna. Bio Process J 2:49–56

    Google Scholar 

  • Ghosh M, Huynh D, Sodhi SS, Sharma N, Kim JH, Kim N, Mongre RK, Park WP, Shin HS, Ko S, Oh S, Choi CW, Oh SJ, Jeong D (2018) Impact of a novel phytase derived from Aspergillus nidulans and expressed in transgenic Lemna minor on the performance, mineralization in bone and phosphorous excretion in laying hens. Pak Vet J 35(3):360–364

    Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155

    CAS  PubMed  Google Scholar 

  • Gifre L, Aris A, Bach A, Garcia-Fruitos E (2017) Trends in recombinant protein use in animal production. Microb Cell Fact 16:40. https://doi.org/10.1186/s12934-017-0654-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Greinacher A, Warkentin TE (2008) The direct thrombin inhibitor hirudin. Thromb Haemost 99(5):819–829

    CAS  PubMed  Google Scholar 

  • Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, Tolstoshev P, Lecocq JP (1986) Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis. Proc Natl Acad Sci USA 83(4):1084–1088

    CAS  PubMed  PubMed Central  Google Scholar 

  • Heenatigala PPM, Sun Z, Yang J, Zhao X, Hou H (2020) Expression of LamB vaccine antigen in Wolffia globosa (duckweed) against fish vibriosis. Front Immunol 11:1857. https://doi.org/10.3389/fimmu.2020.01857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Holaskova E, Galuszka P, Frebort I, Oz MT (2015) Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology. Biotechnol Adv 33(6):1005–1023

    CAS  PubMed  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2(4):208–218

    CAS  Google Scholar 

  • Hu Z, Zhang N, Gu F, Li Y, Deng X, Chen G (2009) Expression, purification and characterization of recombinant targeting bifunctional hirudin in Pichia pastoris. Afr J Biotech 8(20):5571–5577

    CAS  Google Scholar 

  • Karg SR, Kallio PT (2009) The production of biopharmaceuticals in plant systems. Biotechnol Adv 27(6):879–894

    CAS  PubMed  Google Scholar 

  • Khvatkov PA, Chernobrovkina MA, Sinyov VV, Dolgov SV (2013) Study on conditions for Wolffia arrhiza (L.) Horkel ex Wimm submerged culturing in a modified bioreactor. Biotechnology Moscow 6:51–56 (in Russian with English abstract)

    Google Scholar 

  • Khvatkov P, Chernobrovkina M, Okuneva A, Pushin A, Dolgov S (2015a) Transformation of Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss Org Cult 123:299–307

    CAS  Google Scholar 

  • Khvatkov P, Chernobrovkina M, Okuneva A, Shvedova A, Chaban I, Dolgov S (2015b) Callus induction and regeneration in Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss Org Cult 120:263–273

    CAS  Google Scholar 

  • Khvatkov P, Firsov A, Shvedova A, Shaloiko L, Kozlov O, Chernobrovkina M, Pushin A, Tarasenko I, Chaban I, Dolgov S (2018) Development of Wolffia arrhiza as a producer for recombinant human granulocyte colony-stimulating factor. Front Chem 6:304

    PubMed  PubMed Central  Google Scholar 

  • Khvatkov P, Chernobrovkina M, Okuneva A, Dolgov S (2019) Creation of culture media for efficient duckweeds micropropagation (Wolffia arrhiza and Lemna minor) using artificial mathematical optimization models. Plant Cell Tiss Org Cult 136:85–100

    Google Scholar 

  • Kim CH, Seo HW, Kim HY, Sohn JH, Choi ES, Rhee SK (1998) Production of recombinant hirudin in Hansenula polymorpha: variation of gene expression level depends on methanol oxidase and fermentation strategies. J Ind Microbiol Biotechnol 21(1):1–5

    Google Scholar 

  • Ko SM, Sun HJ, Oh MJ, Song IJ, Kim MJ, Sin HS, Goh CH, Kim YW, Lim PO, Lee HY, Kim SW (2011) Expression of the protective antigen for PEDV in transgenic duckweed Lemna minor. Hort Environ Biotechnol 52(5):511–515

    CAS  Google Scholar 

  • Kozlov ON, Mitiouchkina TY, Tarasenko IV, Shaloiko LA, Firsov AP, Dolgov SV (2019) Agrobacterium-mediated transformation of Lemna minor L with hirudin and β-glucuronidase genes. Appl Biochem Microbiol 55(8):805–815

    CAS  Google Scholar 

  • Kruse C, Boehm R, Veste D, Barth S, Schnabl H (2001) Transient transformation of Wolffia columbiana by particle bombardment. AquatBot 72:175–181

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  PubMed  Google Scholar 

  • Landolt E (1986) Biosystematic investigations in the family of duckweeds (Lemnaceae). The family of Lemnaceaea monographic study, Vol. l. Veroffentlichungen des Geobotanischen In-stitutes der E. T. H., Stiftung Rubel, Zurich, Switzerland, 2:638

  • Liu Y, Wang Y, Xu S, Tang X, Zhao J, Yu C, He G, Xu H, Wang S, Tang Y, Fu C, Ma Y, Zhou G (2019) Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis. Plant Biotechnol J 17:2143–2152

    PubMed  PubMed Central  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275

    CAS  PubMed  Google Scholar 

  • Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. BioMed Res Int. https://doi.org/10.1155/2014/136419

    Article  PubMed  PubMed Central  Google Scholar 

  • Michael TP, Ernst E, Hartwick N, Chu P, Bryant D, Gilbert S, Ortleb S, Baggs EL, Sree KS, Appenroth KJ, Fuchs J, Jupe F, Sandoval JP, Krasileva KV, Borisjuk L, Mockler TC, Ecker JR, Martienssen RA, Lam E (2021) Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control. Genome Res 31:225–238

    PubMed Central  Google Scholar 

  • Moloney MM (1995) “Molecular farming” in plants: achievements and prospects. Biotechnol Eng 9:3–9

    Google Scholar 

  • Moon HK, Stomp AM (1997) Effect of medium components and light on callus induction, growth and frond regeneration in Lemna gibba (duckweed) in vitro. Cell Dev Biol 33:20–25

    Google Scholar 

  • Morassutti C, De Amicis F, Skerlavaj B, Zanettib M, Marchetti S (2002) Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system. FEBS Lett 519:141–146

    CAS  PubMed  Google Scholar 

  • Müller C, Mescke K, Liebig S, Mahfoud H, Lemke S, Hildebrandt JP (2016) More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech Hirudo medicinalis. Mol Genet Genomics 291(1):227–240

    PubMed  Google Scholar 

  • Nguyen LV, Cox KM, Ke JS, Peele CG, Dickey LF (2012) Genetic engineering of a Lemna isoleucine auxotroph. Transgenic Res 21(5):1071–1083

    CAS  PubMed  Google Scholar 

  • Niazi SK, Brown JL (2015) Fundamentals of modern bioprocessing. CRC Press, Boca Ratin, p 768

    Google Scholar 

  • Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29(2):210–222

    PubMed  Google Scholar 

  • Parmenter DL, Boothe JG, van Rooijen GJ, Yeung EC, Moloney MM (1995) Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29(6):1167–1180

    CAS  PubMed  Google Scholar 

  • Peterson EJR, Ma S, Sherman DR, Baliga NS (2016) Network analysis identifies Rv0324 and Rv0880 as regulators of bedaquiline tolerance in Mycobacterium tuberculosis. Nat Microbiol. https://doi.org/10.1038/nmicrobiol.2016.78

    Article  PubMed  PubMed Central  Google Scholar 

  • Quétier F (2016) The CRISPR-Cas9 technology: Closer to the ultimate toolkit for targeted genome editing. Plant Sci 242:65–76

    PubMed  Google Scholar 

  • Rader RA, Langer ES (2015) Biopharmaceutical manufacturing: historical and future trends in titers, yields, and efficiency in commercial-scale bioprocessing. BioProcess J 13(4):47–54

    Google Scholar 

  • Radzio R, Kuck U (1997) Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum. Appl Microbiol Biotechnol 48(1):58–65

    CAS  PubMed  Google Scholar 

  • Reinard T, Londenberg A, Brychcy M, Lühmann K, Behrendt G, Wichmann M (2020). In: Fourounjian P, Wang W (eds) Cao X. The duckweed genomes. Compendium of plant genomes. Springer, Cham. https://doi.org/10.1007/978-3-030-11045-1_17

    Chapter  Google Scholar 

  • Rival S, Wisniewski JP, Langlais A, Kaplan H, Freyssinet G, Vancanneyt G, Vunsh R, Perl A, Edelman M (2008) Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res 17(4):503–513

    CAS  PubMed  Google Scholar 

  • Rosenfeld SA, Nadeau D, Tirado J, Hollis GF, Knabb RM, Jia S (1996) Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 8(4):476–482

    CAS  PubMed  Google Scholar 

  • Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204

    CAS  Google Scholar 

  • Sree KS, Maheshwari SC, Boka K, Khurana J, Keresztes A, Appenroth K-J (2015a) The duckweed Wolffia microscopica: aunique aquatic monocot. Flora 210:31–39

    Google Scholar 

  • Sree KS, Sudakaran S, Appenroth K-J (2015b) How fast can angiosperms grow? Species and clonal diversity of growth rates in the genus Wolffia (Lemnaceae). Acta Physiol Plant 37:204

    Google Scholar 

  • Stomp AM (2005) The duckweeds: a valuable plant for biomanufacturing. Biotechnol Annu Rev 11:69–99

    CAS  PubMed  Google Scholar 

  • Strasser R (2016) Plant protein glycosylation. Glycobiology 26(9):926–939

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sun Y, Cheng JJ, Himmel ME, Skory CD, Adney WS, Thomas SR, Tisserat B, Nishimura Y, Yamamoto YT (2007) Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627. Biores Technol 98:2866–2872

    CAS  Google Scholar 

  • Sysuev BB, Pokrovskaya JS (2015) Recombinant microorganisms and cell culture in the technology of protein preparations. Dev Registr Drugs 4:96–109

    Google Scholar 

  • Thu PTL, Anh NH, Huong PT, Hoa NT, Ham LH (2010) Improvement of transformation procedure into duckweed (Wolffia sp.) via Agrobacterium tumefaciens. Tạpchí Côngnghệ Sinhhọc 8:53–60 (in Vietnamese with English abstract)

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS 76(9):4350–4354

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tschofen M, Knopp D, Hood E, Stoger E (2016) Plant molecular farming: much more than medicines. Annu Rev Anal Chem 9:271–294

    Google Scholar 

  • Walsh G (2018) Biopharmaceutical benchmarks 2018. Nat Biotechnol 36(12):1136–1145

    CAS  PubMed  Google Scholar 

  • Wayne AP, Thorne RF (1984) The genus Wolffia (Lemnaceae) in California. Madroño 31(3):171–179

    Google Scholar 

  • Whitelam GC (1995) The production of recombinant proteins in plants. J Sci Food Agric 68:1–9

    CAS  Google Scholar 

  • Wolff P (1992) Les lentilles d'eau de l'Alsace. – Bull. Assoc. Amis Jard.bot. Col de Saveme, 60e anniversaire 1932/1992, pp 25–33. (in French)

  • Yang J, Hu S, Li G, Khan S, Kumar S, Yao L, Duan P, Hou H (2020) Transformation development in duckweeds. In: Cao X, Fourounjian P, Wang W (eds) The duckweed genomes Compendium of plant genomes. Springer, Cham. https://doi.org/10.1007/978-3-030-11045-1_15

    Chapter  Google Scholar 

  • Yarbakht M, Jalali-Javaran M, Nikkhah M, Mohebodini M (2015) Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast. Biotechnol Appl Biochem 62(1):55–63

    CAS  PubMed  Google Scholar 

  • Yasuda H, Tada Y, Hayashi Y, Jomori T, Takaiwa F (2005) Expression of the small peptide GLP-1 in transgenic plants. Transgenic Res 14(5):677–684

    CAS  PubMed  Google Scholar 

  • Yen CH, Yang CK, Chen IC, Lin YS, Lin CS, Chu S, Tu CF (2008) Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter. Biotechnol J 3(8):1067–1077

    CAS  PubMed  Google Scholar 

  • Yevtushenko DP, Misra S (2012) Transgenic expression of antimicrobial peptides in plants: strategies for enhanced disease resistance, improved productivity, and production of therapeutics. Small wonders: peptides for disease control. ACS Symp Ser 1095:445–458

    CAS  Google Scholar 

  • Ziegler P, Adelmann K, Zimmer S, Schmidt C, Appenroth K-J (2015) Relative in vitro growth rates of duckweeds (Lemnaceae)—the most rapidly growing higher plants. Plant Biol 17(1):33–41

    PubMed  Google Scholar 

Download references

Acknowledgements

This research was carried out using the unique scientific facilities "PHYTOBIOGEN" of the «NBS-NSC» RAS (reg. no. 669802; Yalta, Russia) and «PHYTOTRON» reg. #2–2.9 of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS (reg. no 73597; Pushchino, Russia).

Funding

The work was carried out with the support of a grant from the Russian Science Foundation (contract no. 19-74-00010).

Author information

Authors and Affiliations

  1. All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia

    Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Mariya Chernobrovkina, Alexander Pushin & Sergey Dolgov

  2. Nikita Botanical Gardens, Yalta, Russia

    Pavel Khvatkov & Sergey Dolgov

  3. Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Russia

    Alexsey Firsov, Oleg Kozlov, Alexander Pushin, Lyubov Shaloiko & Sergey Dolgov

Authors
  1. Pavel Khvatkov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexsey Firsov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anastasiya Shvedova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Oleg Kozlov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mariya Chernobrovkina
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexander Pushin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lyubov Shaloiko
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sergey Dolgov
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The authors have made the following declarations regarding their contributions: Conceived and designed the experiments: PK, AF, MC, SD. Performed the transformation: PK, AS, MC. PCR and Southern blot analysis: PK, AS, AP. Analysis of hirudin expression AF, OK, LS. Contributed reagents/materials: SD. Contributed to the writing of the manuscript: PK, AF, MC, AP, SD.

Corresponding author

Correspondence to Pavel Khvatkov.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khvatkov, P., Firsov, A., Shvedova, A. et al. Wolffia arrhiza as a promising producer of recombinant hirudin. 3 Biotech 11, 209 (2021). https://doi.org/10.1007/s13205-021-02762-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-021-02762-3

Keywords

Search

Navigation