Skip to main content

Recombinant Protein Production and Purification Using Eukaryotic Cell Factories

  • Protocol
  • First Online:
Biofuels and Biodiesel

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2290))

  • 1076 Accesses

Abstract

Cloning proteins enables their production and characterization for further studies. This requires inserting the gene of the studied protein to be inserted in a vector, which then will be transformed to the host cell used as “factory.” Consequently, the “biomass” of host cells will be produced using bioreactors. Here we describe the production of Rhizomucor miehei lipase (RML) by cloning the corresponding genes in the yeast Pichia pastoris. This enzyme is used as a biocatalyst for biofuel production. The successfully produced recombinant proteins are then purified using ion exchange chromatography.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

  1. Wingfield PT (2015) Overview of the purification of recombinant proteins. Curr Protoc Protein Sci 80:6.1.1–6.1.35. https://doi.org/10.1002/0471140864.ps0601s80

    Article  Google Scholar 

  2. Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 13(2):117–123. https://doi.org/10.1016/s0958-1669(02)00300-2

    Article  CAS  PubMed  Google Scholar 

  3. Kim K, Choe D, Lee DH, Cho BK (2020) Engineering biology to construct microbial chassis for the production of difficult-to-express proteins. Int J Mol Sci 21(3):990. https://doi.org/10.3390/ijms21030990

    Article  CAS  PubMed Central  Google Scholar 

  4. Tripathi NK, Shrivastava A (2019) Recent developments in bioprocessing of recombinant proteins: expression hosts and process development. Front Bioeng Biotechnol 7:420. https://doi.org/10.3389/fbioe.2019.00420

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rodrigues RC, Fernandez-Lafuente R (2010) Lipase from Rhizomucor miehei as a biocatalyst in fats and oils modification. J Mol Catal B Enzym 66(1–2):15–32

    Article  CAS  Google Scholar 

  6. Firdaus MY, Guo Z, Fedosov SN (2016) Development of kinetic model for biodiesel production using liquid lipase as a biocatalyst, esterification step. Biochem Eng J 105:52–61

    Article  CAS  Google Scholar 

  7. Blondeau K, Blaise F, Graille M, Kale SD, Linglin J, Ollivier B, Labarde A, Lazar N, Daverdin G, Balesdent MH, Choi DH, Tyler BM, Rouxel T, van Tilbeurgh H, Fudal I (2015) Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins. Plant J 83(4):610–624. https://doi.org/10.1111/tpj.12913

    Article  CAS  PubMed  Google Scholar 

  8. Yan J, Zheng X, Du L, Li S (2014) Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts. Biotechnol Biofuels 7(1):55. https://doi.org/10.1186/1754-6834-7-55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hu H, Gao J, He J, Yu B, Zheng P, Huang Z, Mao X, Yu J, Han G, Chen D (2013) Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris. PLoS One 8(3):e58393. https://doi.org/10.1371/journal.pone.0058393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Huang J, Xia J, Yang Z, Guan F, Cui D, Guan G, Jiang W, Li Y (2014) Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel. Biotechnol Biofuels 7:111. https://doi.org/10.1186/1754-6834-7-111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Barredo J (2010) Microbial enzymes and biotransformations. Humana Press, Totowa, NJ

    Google Scholar 

  12. Liu Y, Xie W, Yu H (2014) Enhanced activity of Rhizomucor miehei lipase by deglycosylation of its propeptide in Pichia pastoris. Curr Microbiol 68(2):186–191. https://doi.org/10.1007/s00284-013-0460-0

    Article  CAS  PubMed  Google Scholar 

  13. Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A (2012) Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Factories 11:22. https://doi.org/10.1186/1475-2859-11-22

    Article  CAS  Google Scholar 

  14. Wang JR, Li YY, Xu SD, Li P, Liu JS, Liu DN (2013) High-level expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its purification and characterization. Int J Mol Sci 15(1):203–217. https://doi.org/10.3390/ijms15010203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Huang J, Yang Z, Zhu R, Qian X, Wang Y, Li Y, Li J (2018) Efficient heterologous expression of an alkaline lipase and its application in hydrolytic production of free astaxanthin. Biotechnol Biofuels 11:181. https://doi.org/10.1186/s13068-018-1180-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang J, Wang Q, Bu W, Chen L, Yang Z, Zheng W, Li Y, Li J (2019) Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes. Bioengineered 10(1):150–161. https://doi.org/10.1080/21655979.2019.1614422

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang J, Zhao Q, Chen L, Zhang C, Bu W, Zhang X, Zhang K, Yang Z (2020) Improved production of recombinant Rhizomucor miehei lipase by coexpressing protein folding chaperones in Pichia pastoris, which triggered ER stress. Bioengineered 11(1):375–385. https://doi.org/10.1080/21655979.2020.1738127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wongwatanapaiboon J, Malilas W, Ruangchainikom C, Thummadetsak G, Chulalaksananukul S, Marty A, Chulalaksananukul W (2016) Overexpression of Fusarium solani lipase in Pichia pastoris and its application in lipid degradation. Biotechnol Biotechnol Equip 30(5):885–893. https://doi.org/10.1080/13102818.2016.1202779

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The research at the author’s laboratory was developed under the auspices of the CNRS, “Centre National du Recherche Scientifique,” Gif-sur-Yvette, France.

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Missoum, A. (2021). Recombinant Protein Production and Purification Using Eukaryotic Cell Factories. In: Basu, C. (eds) Biofuels and Biodiesel. Methods in Molecular Biology, vol 2290. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1323-8_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1323-8_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1322-1

  • Online ISBN: 978-1-0716-1323-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics