Skip to main content
SpringerLink
Log in

TrypZean™: An Animal-Free Alternative to Bovine Trypsin

  • Chapter
  • First Online:
Commercial Plant-Produced Recombinant Protein Products

Part of the book series: Biotechnology in Agriculture and Forestry ((AGRICULTURE,volume 68))

Abstract

Trypsin derived from either porcine or bovine pancreas is widely used in biopharmaceutical manufacturing. This industry is working toward replacing animal source materials with alternatives to prevent contamination of the products with mammalian viruses and human pathogens. Bovine trypsin made in maize seed culminated in a commercial product, TrypZean™, which is a possible replacement product for use in cell culture and viral vaccine manufacturing, as well as in the processing of therapeutic proteins. This chapter describes the development of this product from a plant biotechnology perspective and then discusses how it is being used today. Possible improvements to make this product more cost competitive are discussed.

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

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Allan DS, Strunk D (2012) Regenerative therapy using blood-derived stem cells. Springer, New York

    Book  Google Scholar 

  • Bause E (1983) Structural requirements of N-glycosylation of proteins. Biochem J 209:331–336

    CAS  PubMed Central  PubMed  Google Scholar 

  • Beyene G, Buenrostro-Nava MT, Damaj MB, Gao S-J, Molina J, Mirkov TE (2011) Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator. Plant Cell Rep 30:13–25

    Article  CAS  PubMed  Google Scholar 

  • Bielanski A, Loewen KG, Hare WCD (1988) Inactivation of bovine herpesvirus-1 (BVH-1) from in vitro infected bovine semen. Theriogenology 30:649–657

    Article  CAS  PubMed  Google Scholar 

  • Brown SW, Mehtali M (2010) The avian EB66 cell line, application to vaccines, and therapeutic protein production. PDA J Pharm Sci Technol 64:419–425

    CAS  PubMed  Google Scholar 

  • Carvalho PP, Wu X, Yu G, Dietrich M, Dias IR, Gomes ME, Reis RL, Gimble JM (2011) Use of animal protein-free products for passaging adherent human adipose-derived stromal/stem cells. Cytotherapy 13:594–597

    Article  CAS  PubMed  Google Scholar 

  • Corey DR, Shiau AK, Yang Q, Janowski BA, Craik CS (1993) Trypsin display on the surface of bacteriophage. Gene 128:129–134

    Article  CAS  PubMed  Google Scholar 

  • Craik CS, Largman C, Fletcher T, Roczniak S, Barr PJ, Fletterick R, Rutter WJ (1985) Redesigning trypsin: alteration of substrate specificity. Science 228:291–297

    Article  CAS  PubMed  Google Scholar 

  • Craik CS, Roczniak S, Largman C, Rutter WJ (1987) The catalytic role of the active site aspartic acid in serine proteases. Science 237:909–913

    Article  CAS  PubMed  Google Scholar 

  • Davie EW, Neurath H (1955) Identification of a peptide released during autocatalytic activation of trypsinogen. J Biol Chem 212:515–529

    CAS  PubMed  Google Scholar 

  • Desai PN, Shrivastava N, Padh H (2010) Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv 28:427–435

    Article  CAS  PubMed  Google Scholar 

  • Doran PM (2006) Loss of secreted antibody from transgenic plant tissue cultures due to surface adsorption. J Biotechnol 122:39–54

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Egelkrout E, McGaughey K, Keener T, Ferleman A, Woodard S, Devaiah S, Nikolov Z, Hood EE, Howard J (2013) Enhanced expression levels of cellulase enzymes using multiple transcription units. Bioenergy Res 6:699–710

    Article  CAS  Google Scholar 

  • Evangelista RL, Kusnadi AR, Howard JA, Nikolov ZL (1998) Process and economic evaluation of the extraction and purification of recombinant β-glucuronidase from transgenic corn. Biotechnol Prog 14:607–614

    Article  CAS  PubMed  Google Scholar 

  • Fekete N, Gadelorge M, Furst D, Maurer C, Dausend J, Fleury-Cappellesso S, Mailander V, Lotfi R, Ignatius A, Sensebe L, Bourin P, Schrezenmeier H, Rojewski MT (2012) Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components. Cytotherapy 14:540–554

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Freshney RI (2010) Culture of animal cells: a manual of basic technique and specialized applications. Wiley, Hoboken, NJ

    Book  Google Scholar 

  • Goulet C, Benchabane M, Anguenot R, Brunelle F, Khalf M, Michaud D (2010) A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants. Plant Biotechnol J 8:142–154

    Article  CAS  PubMed  Google Scholar 

  • Hanquier J, Sorlet Y, Desplancq D, Baroche L, Ebtinger M, Lefevre J-F, Pattus F, Hershberger CL, Vertes AA (2003) A single mutation in the activation site of bovine trypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris. Appl Environ Microbiol 69:1108–1113

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hayden CA, Egelkrout EM, Moscoso AM, Enrique C, Keener TK, Jimenez-Flores R, Wong JC, Howard JA (2012) Production of highly concentrated, heat-stable hepatitis B surface antigen in maize. Plant Biotechnol J 10:979–984

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hedstrom L, Szilagyi L, Rutter WJ (1992) Converting trypsin to chymotrypsin: the role of surface loops. Science 255:1249–1253

    Article  CAS  PubMed  Google Scholar 

  • Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422

    Article  CAS  PubMed  Google Scholar 

  • Hennegan K, Yang D, Nguyen D, Wu L, Goding J, Huang J, Guo F, Huang N, Watkins SC (2005) Improvement of human lysozyme expression in transgenic rice grain by combining wheat (Triticum aestivum) puroindoline b and rice (Oryza sativa) Gt1 promoters and signal peptides. Transgenic Res 14:583–592

    Article  CAS  PubMed  Google Scholar 

  • Hohenblum H, Vorauer-Uhl K, Katinger H, Mattanovich D (2004) Bacterial expression and refolding of human trypsinogen. J Biotechnol 109:3–11

    Article  CAS  PubMed  Google Scholar 

  • Hood EE, Woodard SL (2002) Industrial proteins produced from transgenic plants. In: Hood EE, Howard JA (eds) Plants as factories for protein production. Kluwer, Dordrecht, pp 119–135

    Chapter  Google Scholar 

  • Hood EE, Love R, Lane J, Bray J, Clough R, Pappu K, Drees C, Hood KR, Yoon S, Ahmad A (2007) Subcellular targeting is a key condition for the high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J 5:709–719

    Article  CAS  PubMed  Google Scholar 

  • Hood EE, Devaiah SP, Fake G, Egelkrout E, Teoh TK, Requesens DV, Hayden C, Hood KR, Pappu KM, Carroll J, Howard JA (2012) Manipulating corn germplasm to increase recombinant protein accumulation. Plant Biotechnol J 10:20–30

    Article  CAS  PubMed  Google Scholar 

  • Howard JA, Hood EE (2000) Commercial production of proteases in plants. US 6,087,558 & US 7,049,484 B2

    Google Scholar 

  • Huang J, Nandi S, Wu L, Yalda D, Bartley G, Rodriguez R, Lonnerdal B, Huang N (2002) Expression of natural antimicrobial human lysozyme in rice grains. Mol Breed 10:83–94

    Article  CAS  Google Scholar 

  • Huber R, Bode W (1978) Structural basis of the activation and action of trypsin. Acc Chem Res 11:114–122

    Article  CAS  Google Scholar 

  • Keil B (1971) Trypsin. In: Boyer PD (ed) The enzymes, vol 82. Academic, New York, pp 249–275

    Google Scholar 

  • Kemmler W, Peterson JD, Steiner DF (1971) Studies on the conversion of proinsulin to insulin. I. Conversion in vitro with trypsin and carboxypeptidase B. J Biol Chem 246:6786–6791

    CAS  PubMed  Google Scholar 

  • Kim N-S, Yu H-Y, Chung N-D, Shin Y-J, Kwon T-H, Yang M-S (2011) Production of functional recombinant Bovine trypsin in transgenic rice cell suspension cultures. Protein Expr Purif 76:121–126

    Article  CAS  PubMed  Google Scholar 

  • Kusnadi AR, Nikolov ZL, Howard JA (1997) Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol Bioeng 56:473–484

    Article  CAS  PubMed  Google Scholar 

  • Kwon J-Y, Lee K-H, Cheon S-H, Ryu H-N, Kim SJ, Kim D-I (2012) Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures. Plant Cell Rep 31:551–560

    Article  CAS  PubMed  Google Scholar 

  • Liepnieks JJ, Light A (1974) Preparation of beta-trypsin by affinity chromatography of enterokinase-activated bovine trypsinogen. Anal Biochem 60:395–404

    Article  CAS  PubMed  Google Scholar 

  • Mattson KJ, Devlin BR, Loskutoff NM (2008) Comparison of a recombinant trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos. Theriogenology 69:724–727

    Article  CAS  PubMed  Google Scholar 

  • McClenahan SD, Krause PR, Uhlenhaut C (2011) Molecular and infectivity studies of porcine circovirus in vaccines. Vaccine 29:4745–4753

    Article  CAS  PubMed  Google Scholar 

  • Merten O-W (2002) Virus contamination of cell cultures—a biotechnological view. Cytotechnology 39:91–116

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Muller R, Glaser S, Geipel F, Thalhofer J-P, Rexer B, Schneider C, Ratka M, Ronning S, Eckstein H, Giessel C (2010) Method for producing recombinant trypsin. US 7,666,629

    Google Scholar 

  • Murphy S, Rosli S, Acharya R, Mathias L, Lim R, Wallace E, Jenkin G (2010) Amnion epithelial cell isolation and characterization for clinical use. Curr Protoc Stem Cell Biol 13:1E.6.1–1E.6.25

    Google Scholar 

  • Nikolov Z, Hammes D (2002) Production of recombinant proteins from transgenic crops. In: Hood EE, Howard JA (eds) Plants as factories for protein production. Kluwer, Dordrecht, pp 159–174

    Chapter  Google Scholar 

  • Pau MG, UytdeHaag AG (2003) Use of recombinant trypsin for vaccine production. WO 03/076462 A1

    Google Scholar 

  • Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F (2001) The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine 19:2716–2721

    Article  CAS  PubMed  Google Scholar 

  • Peterson FC, Gordon NC, Gettins PGW (2001) High-level bacterial expression and 15alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy. Biochemistry 40:6275–6283

    Article  CAS  PubMed  Google Scholar 

  • Polak-Vogelzang AA, Angulo AF, Brugman J, Reijgers R (1990) Survival of Mycoplasma hyorhinis in trypsin solutions. Biologicals 18:97–101

    Article  CAS  PubMed  Google Scholar 

  • Rawlings ND, Barrett AJ (1994) Families of serine peptidases. In: Barrett AJ (ed) Methods in enzymology, vol 244. Academic, San Diego, CA, pp 19–61

    Google Scholar 

  • Ray K, Jalili PR (2011) Characterization of TrypZean: a plant based alternative to bovine-derived trypsin. Biopharm Int 24(10):44–48

    CAS  Google Scholar 

  • Rogers JC (1985) Two barley alpha-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260:3731–3738

    CAS  PubMed  Google Scholar 

  • Rourou S, van de Ark A, van de Velden T (2009) Development of an animal-component free medium for Vero cells culture. Biotechnol Prog 25:1752–1761

    CAS  PubMed  Google Scholar 

  • Schroeder DD, Shaw E (1968) Chromatography of trypsin and its derivatives. Characterization of a new active form of bovine trypsin. J Biol Chem 243:2943–2949

    CAS  PubMed  Google Scholar 

  • Seidel GE Jr, Turk ML, Gordy PW, Bowen RA (2007) Recombinant bovine trypsin made in maize inactivates bovine herpes virus-1 absorbed to the bovine zona pellucida. Reprod Fertil Dev 19:236 [abstract]

    Article  Google Scholar 

  • Shigemitsu T, Ozaki S, Saito Y, Kuroda M, Morita S, Satoh S, Masumura T (2012) Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins. Plant Cell Rep 31:539–549

    Article  CAS  PubMed  Google Scholar 

  • Silva FJ, Gonzalez R (2013) Umbilical cord lining stem cells and methods and material for isolating and culturing same. US2013/0065302

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Streatfield SJ, Bray J, Love RT, Horn ME, Lane JR, Drees CF, Egelkrout EM, Howard JA (2010) Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production. GM Crops 1:162–172

    Article  PubMed  Google Scholar 

  • Stryer L (1995) Biochemistry, 4th edn. WH Freeman, New York

    Google Scholar 

  • Szilagyi L, Kenesi E, Katona G, Kaslik G, Juhasz G, Graf L (2001) Comparative in vitro studies on native and recombinant human cationic trypsins. J Biol Chem 276:24574–24580

    Article  CAS  PubMed  Google Scholar 

  • Vasquez JR, Evnin LB, Higaki JN, Craik CS (1989) An expression system for trypsin. J Cell Biochem 39:265–276

    Article  CAS  PubMed  Google Scholar 

  • Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, Delaney DE, McComas-Wagner JM, Mallubhotla HD, Hood EE, Dangott LJ, Tichy SE, Howard JA (2003) Maize-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol Appl Biochem 38:123–130

    Article  CAS  PubMed  Google Scholar 

  • Yee L, Blanch HW (1993) Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli. Biotechnol Bioeng 41:781–790

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Huang RY-C, Jalili PR, Irungu JW, Nicol GR, Ray KB, Rohrs HW, Gross ML (2010) Improved mass spectrometric characterization of protein glycosylation reveals unusual glycosylation of maize-derived bovine trypsin. Anal Chem 15:10095–10101

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lasergen Inc., 8052 El Rio Street, Houston, TX, 77054, USA

    Aparna Krishnan

  2. Kalon Biotherapeutics, 100 Discovery Drive, Suite 200, College Station, TX, 77845, USA

    Susan L. Woodard

Authors
  1. Aparna Krishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan L. Woodard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susan L. Woodard .

Editor information

Editors and Affiliations

  1. Cal Poly Technology Park, Applied Biotechnology Institute, San Luis Obispo, California, USA

    John A. Howard

  2. Arkansas Biosciences Institute, Arkansas State University, Jonesboro, Arkansas, USA

    Elizabeth E. Hood

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Krishnan, A., Woodard, S.L. (2014). TrypZean™: An Animal-Free Alternative to Bovine Trypsin. In: Howard, J., Hood, E. (eds) Commercial Plant-Produced Recombinant Protein Products. Biotechnology in Agriculture and Forestry, vol 68. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-43836-7_4

Download citation

Publish with us

Policies and ethics

Search

Navigation