Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3145–3158 | Cite as

Heterologous expression of rTsHyal-1: the first recombinant hyaluronidase of scorpion venom produced in Pichia pastoris system

  • Fernanda Gobbi Amorim
  • Johara Boldrini-França
  • Karla de Castro Figueiredo Bordon
  • Iara Aimê Cardoso
  • Edwin De Pauw
  • Loïc Quinton
  • Simone Kashima
  • Eliane Candiani Arantes
Biotechnologically relevant enzymes and proteins


In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies. Therefore, this study produced a recombinant hyaluronidase from Tityus serrulatus scorpion venom, designated as rTsHyal-1, in the Pichia pastoris system. Thus, a gene for TsHyal-1 (gb|KF623285.1) was synthesized and cloned into the pPICZαA vector (GenScript Corporation) for heterologous expression in P. pastoris. rTsHyal-1 was expressed in laboratorial scale in a buffered minimal medium containing methanol (BMM) for 96 h with daily addition of methanol. Expression of rTsHyal-1 resulted in a total protein yield of 0.266 mg/mL. rTsHyal-1 partially purified through cation exchange chromatography presented a specific activity of 1097 TRU/mg, against 838 TRU/mg for the final expressed material, representing a 1.31-fold purification. rTsHyal-1 has molecular mass of 49.5 kDa, and treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, de novo sequencing of rTsHyal-1, performed in MALDI-TOF and Q Exactive Orbitrap MS, resulted in 46.8% of protein sequence coverage. rTsHyal-1 presents the highest substrate specificity to hyaluronan followed by chondroitin-6-sulfate, chondroitin-4-sulfate, and dermatan sulfate and showed an optimum activity at pH 6.0 and 40 °C. These results validate the biotechnological process for the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpion venoms expressed in the P. pastoris system with preserved enzyme activity.


Tityus serrulatus Scorpion venom Hyaluronidase Heterologous expression Pichia pastoris 


Compliance with ethical standards

Ethical statement approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of interest

The authors declare that they have no conflict of interest.


  1. Amorim FG, Morandi-Filho R, Fujimura PT, Ueira-Vieira C, Sampaio SV (2017) New findings from the first transcriptome of the Bothrops moojeni snake venom gland. Toxicon 140:105–117. CrossRefPubMedGoogle Scholar
  2. Bakke M, Kamei J, Obata A (2011) Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp. FEBS Lett 585(1):115–120. CrossRefPubMedGoogle Scholar
  3. Batista CV, Román-González SA, Salas-Castillo SP, Zamudio FZ, Gómez-Lagunas F, Possani LD (2007) Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species. Comp Biochem Physiol C Toxicol Pharmacol 146(1-2):147-157.
  4. Berry AM, Lock RA, Thomas SM, Rajan DP, Hansman D, Paton JC (1994) Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli. Infect Immun 62(3):1101–1108PubMedPubMedCentralGoogle Scholar
  5. Bordon KC, Perino MG, Giglio JR, Arantes EC (2012) Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom. Biochimie 94(12):2740–2748. CrossRefPubMedGoogle Scholar
  6. Bordon KC, Wiezel GA, Amorim FG, Arantes EC (2015) Arthropod venom hyaluronidases: biochemical properties and potential applications in medicine and biotechnology. J Venom Anim Toxins Incl Trop Dis 21(1):43. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Brondyk WH (2009) Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol 463:131–147. CrossRefPubMedGoogle Scholar
  8. Calvete JJ (2017) Venomics: integrative venom proteomics and beyond. Biochem J 474(5):611–634. CrossRefPubMedGoogle Scholar
  9. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66. CrossRefPubMedGoogle Scholar
  10. Cevallos MA, Navarro-Duque C, Varela-Julia M, Alagon AC (1992) Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Toxicon 30(8):925–930. CrossRefPubMedGoogle Scholar
  11. Chen KJ, Sabrina S, El-Safory NS, Lee GC, Lee CK (2016) Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris. J Biosci Bioeng 122(6):673–678. CrossRefPubMedGoogle Scholar
  12. Clement H, Olvera A, Rodríguez M, Zamudio F, Palomares LA, Possani LD, Odell GV, Alagón A, Sánchez-López R (2012) Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom. Toxicon 60(7):1223–1227. CrossRefPubMedGoogle Scholar
  13. Cologna CT, Marcussi S, Giglio JR, Soares AM, Arantes EC (2009) Tityus serrulatus scorpion venom and toxins: an overview. Protein Pept Lett 16(8):920–932. CrossRefPubMedGoogle Scholar
  14. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16(1):23–52. CrossRefPubMedGoogle Scholar
  15. Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18(2):119–138. CrossRefPubMedGoogle Scholar
  16. Feng L, Gao R, Gopalakrishnakone P (2008) Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi. Comp Biochem Physiol C Toxicol Pharmacol 148(3):250–257. CrossRefPubMedGoogle Scholar
  17. Ferrer VP, de Mari TL, Gremski LH, Trevisan Silva D, da Silveira RB, Gremski W, Chaim OM, Senff-Ribeiro A, Nader HB, Veiga SS (2013) A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich’s hyaluronidase): from cloning to functional characterization. PLoS Negl Trop Dis 7(5):e2206. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Frost GI, Csóka AB, Wong T, Stern R, Csóka TB (1997) Purification, cloning, and expression of human plasma hyaluronidase. Biochem Biophys Res Commun 236(1):10–15. CrossRefPubMedGoogle Scholar
  19. Gao MJ, Zhan XB, Gao P, Zhang X, Dong SJ, Li Z, Shi ZP, Lin CC (2015) Improving performance and operational stability of porcine interferon-α production by Pichia pastoris with combinational induction strategy of low temperature and methanol/sorbitol co-feeding. Appl Biochem Biotechnol 176(2):493–504. CrossRefPubMedGoogle Scholar
  20. Gmachl M, Kreil G (1993) Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc Natl Acad Sci U S A 90(8):3569–3573. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Goto Y, Niwa Y, Suzuki T, Uematsu S, Dohmae N, Simizu S (2014) N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1. FEBS Open Bio 4(1):554–559. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Heimo H, Palmu K, Suominen I (1997) Expression in Pichia pastoris and purification of Aspergillus awamori glucoamylase catalytic domain. Protein Expr Purif 10(1):70–79. CrossRefPubMedGoogle Scholar
  23. Hofinger ES, Spickenreither M, Oschmann J, Bernhardt G, Rudolph R, Buschauer A (2007) Recombinant human hyaluronidase Hyal-1: insect cells versus Escherichia coli as expression system and identification of low molecular weight inhibitors. Glycobiology 17(4):444–453. CrossRefPubMedGoogle Scholar
  24. Horta CC, Magalhães BF, Oliveira-Mendes BB, do Carmo AO, Duarte CG, Felicori LF, Machado-de-Ávila RA, Chávez-Olórtegui C, Kalapothakis E (2014) Molecular, immunological, and biological characterization of Tityus serrulatus venom hyaluronidase: new insights into its role in envenomation. PLoS Negl Trop Dis 8(2):e2693. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jin P, Kang Z, Zhang N, Du G, Chen J (2014) High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers. Sci Rep 4(1):4471. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jung Y, Jung MY, Park JH, Jung GC, Hong YS, Yeom CH, Lee S (2010) Production of human hyaluronidase in a plant-derived protein expression system: plant-based transient production of active human hyaluronidase. Protein Expr Purif 74(2):181–188. CrossRefPubMedGoogle Scholar
  27. Justo Jacomini DL, Gomes Moreira SM, Campos Pereira FD, Zollner RL, Brochetto Braga MR (2014) Reactivity of IgE to the allergen hyaluronidase from Polybia paulista (Hymenoptera, Vespidae) venom. Toxicon 82:104–111. CrossRefPubMedGoogle Scholar
  28. Kang Z, Zhang N, Zhang Y (2016) Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris. Appl Microbiol Biotechnol 100(2):707–717. CrossRefPubMedGoogle Scholar
  29. King TP, Lu G, Gonzalez M, Qian N, Soldatova L (1996) Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy. J Allergy Clin Immunol 98(3):588–600. CrossRefPubMedGoogle Scholar
  30. Kuhn-Nentwig L (2003) Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci 60(12):2651–2668. CrossRefPubMedGoogle Scholar
  31. Laemmli UK, Beguin F, Gujer-Kellenberger G (1970) A factor preventing the major head protein of bacteriophage T4 from random aggregation. J Mol Biol 47(1):69–85. CrossRefPubMedGoogle Scholar
  32. Lambers H, Piessens S, Bloem A, Pronk H, Finkel P (2006) Natural skin surface pH is on average below 5, which is beneficial for its resident flora. Int J Cosmet Sci 28(5):359–370. CrossRefPubMedGoogle Scholar
  33. Li H, Yang J, Chen Y, Guan L, Du L, Guo Y, Wang W, Wang L, Jiang C, Li X (2014) Expression of a functional recombinant oleosin-human hyaluronidase hPH-20 fusion in Arabidopsis thaliana. Protein Expr Purif 103:23–27. CrossRefPubMedGoogle Scholar
  34. Li MW, Yudin AI, Robertson KR, Cherr GN, Overstreet JW (2002) Importance of glycosylation and disulfide bonds in hyaluronidase activity of macaque sperm surface PH-20. J Androl 23(2):211–219PubMedGoogle Scholar
  35. Lu G, Kochoumian L, King TP (1995) Sequence identity and antigenic cross-reactivity of white face hornet venom allergen, also a hyaluronidase, with other proteins. J Biol Chem 270(9):4457–4465. CrossRefPubMedGoogle Scholar
  36. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249–270. CrossRefPubMedGoogle Scholar
  37. Mechref Y (2012) Use of CID/ETD mass spectrometry to analyze glycopeptides. Curr Protoc Protein Sci Chapter 12:Unit 12.11.11-11.
  38. Mergulhão FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23(3):177–202. CrossRefPubMedGoogle Scholar
  39. Morelle W, Michalski JC (2007) Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2(7):1585–1602. CrossRefPubMedGoogle Scholar
  40. Morey SS, Kiran KM, Gadag JR (2006) Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom. Toxicon 47(2):188–195. CrossRefPubMedGoogle Scholar
  41. Nair RB, Kurup PA (1975) Investigations on the venom of the South Indian scorpion Heterometrus scaber. Biochim Biophys Acta 381(1):165–174. CrossRefPubMedGoogle Scholar
  42. Ng HC, Ranganathan S, Chua KL, Khoo HE (2005) Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida). Gene 346:71–81. CrossRefPubMedGoogle Scholar
  43. Ohman H, Vahlquist A (1998) The pH gradient over the stratum corneum differs in X-linked recessive and autosomal dominant ichthyosis: a clue to the molecular origin of the "acid skin mantle"? J Invest Dermatol 111(4):674–677. CrossRefPubMedGoogle Scholar
  44. Pessini AC, Takao TT, Cavalheiro EC, Vichnewski W, Sampaio SV, Giglio JR, Arantes EC (2001) A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids. Toxicon 39(10):1495–1504. CrossRefPubMedGoogle Scholar
  45. Pucca MB, Amorim FG, Cerni FA, Bordon KC, Cardoso IA, Anjolette FA, Arantes EC (2014) Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity. Toxicon 90:326–336. CrossRefPubMedGoogle Scholar
  46. Pucca MB, Cerni FA, Pinheiro Junior EL, Bordon KC, Amorim FG, Cordeiro FA, Longhim HT, Cremonez CM, Oliveira GH, Arantes EC (2015) Tityus serrulatus venom—a lethal cocktail. Toxicon 108:272–284. CrossRefPubMedGoogle Scholar
  47. Pukrittayakamee S, Warrell DA, Desakorn V, McMichael AJ, White NJ, Bunnag D (1988) The hyaluronidase activities of some Southeast Asian snake venoms. Toxicon 26(7):629–637. CrossRefPubMedGoogle Scholar
  48. Ramanaiah M, Parthasarathy PR, Venkaiah B (1990) Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom. Biochem Int 20(2):301–310PubMedGoogle Scholar
  49. Reitinger S, Boroviak T, Laschober GT, Fehrer C, Müllegger J, Lindner H, Lepperdinger G (2008) High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris. Protein Expr Purif 57(2):226–233. CrossRefPubMedGoogle Scholar
  50. Rodríguez-Ravelo R, Coronas FI, Zamudio FZ, González-Morales L, López GE, Urquiola AR, Possani LD (2013) The Cuban scorpion Rhopalurus junceus (Scorpiones, Buthidae): component variations in venom samples collected in different geographical areas. J Venom Anim Toxins Incl Trop Dis 19(1):13. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Scopes RK (1974) Measurement of protein by spectrophotometry at 205 nm. Anal Biochem 59(1):277–282. CrossRefPubMedGoogle Scholar
  52. Skov LK, Seppala U, Coen JJF, Crickmore N, King TP, Monsalve R, Kastrup JS, Spangfort MD, Gajhede M (2006) Structure of recombinant Ves v 2 at 2.0 angstrom resolution: structural analysis of an allergenic hyaluronidase from wasp venom. Acta Crystallogr D Biol Crystallogr 62(6):595–604. CrossRefPubMedGoogle Scholar
  53. Soldatova LN, Crameri R, Gmachl M, Kemeny DM, Schmidt M, Weber M, Mueller UR (1998) Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli. J Allergy Clin Immunol 101(5):691–698. CrossRefPubMedGoogle Scholar
  54. Soldatova LN, Tsai C, Dobrovolskaia E, Marković-Housley Z, Slater JE (2007) Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells. Allergy Asthma Proc 28(2):210–215. CrossRefPubMedGoogle Scholar
  55. Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay J-T, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190(1):55–62Google Scholar
  56. Tan CH, Fung SY, Yap MK, Leong PK, Liew JL, Tan NH (2016) Unveiling the elusive and exotic: venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps). J Proteome 132:1–12. CrossRefGoogle Scholar
  57. Venancio EJ, Portaro FC, Kuniyoshi AK, Carvalho DC, Pidde-Queiroz G, Tambourgi DV (2013) Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms. Toxicon 69:180–190. CrossRefPubMedGoogle Scholar
  58. Wiezel GA, Dos Santos PK, Cordeiro FA, Bordon KC, Selistre-de-Araújo HS, Ueberheide B, Arantes EC (2015) Identification of hyaluronidase and phospholipase B in Lachesis muta rhombeata venom. Toxicon 107(Pt B):359–368. CrossRefPubMedGoogle Scholar
  59. Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 80:6.1.1–6.135. CrossRefGoogle Scholar
  60. Wohlrab J, Wohlrab D, Wohlrab L, Wohlrab C, Wohlrab A (2014) Use of hyaluronidase for pharmacokinetic increase in bioavailability of intracutaneously applied substances. Skin Pharmacol Physiol 27(5):276–282. CrossRefPubMedGoogle Scholar
  61. Xia X, Liu R, Li Y, Xue S, Liu Q, Jiang X, Zhang W, Ding K (2014) Cloning and molecular characterization of scorpion Buthus martensi venom hyaluronidases: a novel full-length and diversiform noncoding isoforms. Gene 547(2):338–345. CrossRefPubMedGoogle Scholar
  62. Zaia J (2004) Mass spectrometry of oligosaccharides. Mass Spectrom Rev 23(3):161–227. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Fernanda Gobbi Amorim
    • 1
  • Johara Boldrini-França
    • 1
  • Karla de Castro Figueiredo Bordon
    • 1
  • Iara Aimê Cardoso
    • 1
  • Edwin De Pauw
    • 2
  • Loïc Quinton
    • 2
  • Simone Kashima
    • 3
  • Eliane Candiani Arantes
    • 1
  1. 1.School of Pharmaceutical Sciences of Ribeirão Preto, Department of Physics and ChemistryUniversity of São PauloRibeirão PretoBrazil
  2. 2.Department of ChemistryUniversity of LiègeLiègeBelgium
  3. 3.Ribeirão Preto Medical School, Regional Center for Hemotherapy of Ribeirão PretoUniversity of São PauloSão PauloBrazil

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