Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Secreted production of an elastin-like polypeptide by Pichia pastoris

Abstract

Elastin-like polypeptides (ELPs) are biocompatible designer polypeptides with inverse temperature transition behavior in solution. They have a wide variety of possible applications and a potential medical importance. Currently, production of ELPs is done at lab scale in Escherichia coli shake flask cultures. With a view to future large scale production, we demonstrate secreted production of ELPs in methanol-induced fed-batch cultures of Pichia pastoris and purification directly from the culture medium. The production of ELPs by P. pastoris proved to be pH dependent within the experimental pH range of pH 3 to 7, as an increasing yield was found in cultures grown at higher pH. Because ELP produced at pH 7 was partly degraded, a pH optimum for production of ELP was found at pH 6 with a yield of 255 mg of purified intact ELP per liter of cell-free medium.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Bedellhogan D, Trackman P, Abrams W, Rosenbloom J, Kagan H (1993) Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase. J Biol Chem 268:10345–10350

  2. Bollok M, Henriksson H, Kallas A, Jahic M, Teeri TT, Enfors SO (2005) Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris. Appl Biochem Biotechnol 126:61–77

  3. Cappello J (1990) The biological production of protein polymers and their use. Trends Biotechnol 8:309–311

  4. Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332. doi:https://doi.org/10.1016/S0958166902003300

  5. Chambers SP, Prior SE, Barstow DA, Minton NP (1988) The pMTL nic - cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149

  6. Chen T-H, Bae Y, Furgeson D (2008) Intelligent biosynthetic nanobiomaterials (IBNs) for hyperthermic gene delivery. Pharmaceutical Res 25:683–691

  7. Chilkoti A, Dreher MR, Meyer DE (2002) Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery. Adv Drug Deliv Rev 54:1093–1111

  8. Chilkoti A, Christensen T, MacKay JA (2006) Stimulus responsive elastin biopolymers: Applications in medicine and biotechnology. Curr Opin Chem Biol 10:652–657. doi:https://doi.org/10.1016/j.cbpa.2006.10.010

  9. Chow D, Nunalee ML, Lim DW, Simnick AJ, Chilkoti A (2008) Peptide-based biopolymers in biomedicine and biotechnology. Mater Sci Eng, R Rep 62:125–155

  10. Christensen T, Trabbic-Carlson K, Liu W, Chilkoti A (2007) Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling. Anal Biochem 360:166–168

  11. Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA (1991) Production of mouse epidermal growth-factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105:205–212

  12. Cox BA, Starcher BC, Urry DW (1974) Coacervation of tropoelastin results in fiber formation. J Biol Chem 249:997–998

  13. Cregg J, Cereghino J, Shi J, Higgins D (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52

  14. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306

  15. Egel-Mitani M, Hansen MT (1987) Nucleotide-sequence of the gene encoding the Saccharomyces kluyveri alpha-mating pheromone. Nucleic Acids Res 15:6303–6303

  16. Files D, Ogawa M, Scaman CH, Baldwin SA (2001) A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C. Enzyme Microb Technol 29:335–340

  17. Gacko M (2000) Elastin: Structure, properties and metabolism. Cell Mol Biol Lett 5:327–348

  18. Ge X, Filipe CDM (2006) Simultaneous phase transition of ELP tagged molecules and free elp: an efficient and reversible capture system. Biomacromolecules 7:2475–2478. doi:https://doi.org/10.1021/bm060507n

  19. Ge X, Yang DSC, Trabbic-Carlson K, Kim B, Chilkoti A, Filipe CDM (2005) Self-cleavable stimulus responsive tags for protein purification without chromatography. J Am Chem Soc 127:11228–11229

  20. Gillies A, Hsii J, Oak S, Wood D (2008) Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags. Biotechnol Bioeng 101:229–240

  21. Guda C, Zhang X, McPherson DT, Xu J, Cherry JH, Urry DW, Daniell H (1995) Hyper expression of an environmentally friendly synthetic polymer gene. Biotechnol Lett 17:745–750

  22. Guda C, Lee SB, Daniell H (2000) Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Rep 19:257

  23. Herzog RW, Singh NK, Urry DW, Daniell H (1997) Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans. Appl Microbiol Biotechnol 47:368–372

  24. Heslot H (1998) Artificial fibrous proteins: a review. Biochimie 80:19–31

  25. Inan M, Chiruvolu V, Eskridge KM, Vlasuk GP, Dickerson K, Brown S, Meagher MM (1999) Optimization of temperature-glycerol-pH conditions for a fed-batch fermentation process for recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (AcAP-5) production by Pichia pastoris. Enzyme Microb Technol 24:438–445

  26. Jahic M, Gustavsson M, Jansen AK, Martinelle M, Enfors SO (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 102:45–53. doi:https://doi.org/10.1016/S0168-1656(03)00003-8

  27. Kreil G (1990) Processing of precursors by dipeptidylaminopeptidases: a case of molecular ticketing. Trends Biochem Sci 15:23

  28. Lee C, Levin A, Branton D (1987) Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 166:308

  29. Li PZ, Anumanthan A, Gao XG, Ilangovan K, Suzara VV, Duzgunes N, Renugopalakrishnan V (2007) Expression of recombinant proteins in Pichia pastoris. Appl Biochem Biotechnol 142:105–124

  30. Matoba S, Fukayama J, Wing RA, Ogrydziak DM (1988) Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica. Mol Cell Biol 8:4904–4916

  31. Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135

  32. McHale MK, Setton LA, Chilkoti A (2005) Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair. Tissue Eng 11:1768–1779

  33. Megeed Z, Cappello J, Ghandehari H (2002) Genetically engineered silk-elastinlike protein polymers for controlled drug delivery. Adv Drug Deliv Rev 54:1075

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

  35. Meyer DE, Chilkoti A (2002) Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 3:357–367. doi:https://doi.org/10.1021/bm015630n

  36. Meyer DE, Chilkoti A (2004) Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides. Biomacromolecules 5:846–851

  37. Mithieux SM, Weiss AS (2005) Elastin. In: Parry AD (ed) Fibrous proteins: coiled-coils, collagen and elastomers. Elsevier Academic, San Diego, pp 437–458

  38. Rincon AC, Molina-Martinez IT, de Las Heras B, Alonso M, Bailez C, Rodriguez-Cabello JC, Herrero-Vanrell R (2006) Biocompatibility of elastin-like polymer poly(VPAGV) microparticles: in vitro and in vivo studies. J Biomed Mat Res Part A 78A:343–351. doi:https://doi.org/10.1002/jbm.a.30702

  39. Rodriguez-Cabello JC, Prieto S, Reguera J, Arias FJ, Ribeiro A (2007) Biofunctional design of elastin-like polymers for advanced applications in nanobiotechnology. J Biomater Sci, Polym Ed 18:269–286. doi:https://doi.org/10.1163/156856207779996904

  40. Simnick AJ, Lim DW, Chow D, Chilkoti A (2007) Biomedical and biotechnological applications of elastin-like polypeptides. Polym Rev 47:121–154

  41. Sinha J, Plantz BA, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-τ. Biotechnol Bioeng 89:102–112. doi:https://doi.org/10.1002/bit.20318

  42. Toonkool P, Weiss AS (2001) Expression of recombinant human tropoelastin in Saccharomyces cerevisiae containing a synthetic gene with a high codon adaptation index coupled to the SUC2 invertase signal sequence. Acta Biotechnol 21:189–193

  43. Trabbic-Carlson K, Setton LA, Chilkoti A (2003) Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides. Biomacromolecules 4:572–580. doi:https://doi.org/10.1021/bm025671z

  44. 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

  45. Urry DW (1976) Molecular mechanisms of elastin coacervation and coacervate calcification. Faraday Discus 205–212

  46. Urry DW (1992) Free energy transduction in polypeptides and proteins based on inverse temperature transitions. Prog Biophys Mol Biol 57:23–57

  47. Urry DW, Long MM, Cox BA, Ohnishi T, Mitchell LW, Jacobs M (1974) Synthetic polypentapeptide of elastin coacervates and forms filamentous aggregates. Biochim Biophys Acta, Protein Struct 371:597–602

  48. Urry DW, Luan CH, Parker TM, Gowda DC, Prasad KU, Reid MC, Safavy A (1991a) Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity. J Am Chem Soc 113:4346–4348

  49. Urry DW, Parker TM, Reid MC, Gowda DC (1991b) 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

  50. Urry DW, Gowda DC, Parker TM, Luan CH, Reid MC, Harris CM, Pattanaik A, Harris RD (1992) Hydrophobicity scale for proteins based on inverse temperature transitions. Biopolymers 32:1243–1250

  51. Wenzel T, Sparbier K, Mieruch T, Kostrzewa M (2006) 2, 5-dihydroxyacetophenone: a matrix for highly sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of proteins using manual and automated preparation techniques. Rapid Commun Mass Spectrom 20:785–789. doi:https://doi.org/10.1002/rcm.2378

  52. Werten MWT, Van den Bosch TJ, Wind RD, Mooibroek H, De Wolf FA (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1087–1096

  53. Werten MWT, Wisselink WH, Jansen-van den Bosch TJ, de Bruin EC, de Wolf FA (2001) Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris. Protein Eng 14:447–454

  54. Werten MWT, Moers APHA, Vong T, Zuilhof H, van Hest JCM, de Wolf FA (2008) Biosynthesis of an amphiphilic silk-like polymer. Biomacromolecules 9:1705–1711

  55. Zhang X, Guda C, Datta R, Dute R, Urry DW, Daniell H (1995) Nuclear expression of an environmentally friendly synthetic protein based polymer gene in tobacco cells. Biotechnol Lett 17:1279–1284

  56. Zhang W, Bevins M, Plantz B, Smith L, Meagher M (2000) Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A. Biotechnol Bioeng 70:1–8

  57. Zhang W, Inan M, Meagher MM (2007) Rational design and optimization of fed-batch and continuous fermentations. In: Cregg J (ed) Pichia protocols, 3rd edn. Humana, Totowa, pp 43–63

  58. Zhao W, Wang JW, Deng RQ, Wang XZ (2008) Scale-up fermentation of recombinant Candida rugosa lipase expressed in Pichia pastoris using the GAP promoter. J Ind Microbiol Biotech 35:189–195

Download references

Acknowledgment

The authors are grateful to Aernout Martens who provided the pMTL23-AIII cloning vector that was used for oligomerization and Antoine Moers for his assistance in the practical work. This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public–private NWO-ACTS program (ACTS = Advanced Chemical Technologies for Sustainability).

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Author information

Correspondence to Frits A. de Wolf.

Rights and permissions

Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Reprints and Permissions

About this article

Cite this article

Schipperus, R., Teeuwen, R.L.M., Werten, M.W.T. et al. Secreted production of an elastin-like polypeptide by Pichia pastoris . Appl Microbiol Biotechnol 85, 293–301 (2009). https://doi.org/10.1007/s00253-009-2082-9

Download citation

Keywords

  • Elastin-like polypeptides
  • Pichia pastoris
  • Secreted expression
  • Inverse transition cycling
  • Heterologous protein expression