Skip to main content
SpringerLink
Log in

Quantification of Recombinant Products in Yeast

  • Protocol
  • First Online:
Recombinant Protein Production in Yeast

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1923))

  • 3077 Accesses

Abstract

Quantification of various proteins expressed in yeast can be performed by different methods. In this respect, classical as well as advanced techniques can be applied, where the analysis of crude supernatants is of special interest in screening but also manufacturing.

The following chapter addresses the analytical background of the introduced methods followed by specific recommendations for the quantification of different products of industrial interest. The method portfolio includes electrophoresis, chromatography, and ELISA as classical techniques, but also biosensor-based, microfluidic and automated, miniaturized methods are introduced. Furthermore, individual strengths and perceived limitations are summarized.

Although prominent examples are described, it should be noted that individual modifications are required according to host and cultivation mode.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
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. Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317. https://doi.org/10.1007/s00253-014-5732-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Liu Z, Tyo KEJ, Martinez JL et al (2012) Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng 109:1259–1268. https://doi.org/10.1002/bit.24409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Weinacker D, Rabert C, Zepeda AB et al (2013) Applications of recombinant Pichia pastoris in the healthcare industry. Brazilian J Microbiol 44:1043–1048. https://doi.org/10.1590/S1517-83822013000400004

    Article  Google Scholar 

  4. Porro D, Sauer M, Branduardi P, Mattanovich D (2005) Recombinant protein production in yeasts. Mol Biotechnol 31:1–15

    Article  Google Scholar 

  5. Nielsen KH (2014) Protein expression-yeast. Methods Enzymol 536:133–147. https://doi.org/10.1016/B978-0-12-420070-8.00012-X

    Article  CAS  PubMed  Google Scholar 

  6. Garfin DE (2009) Chapter 29: One-dimensional gel electrophoresis. Methods Enzymol 463:497–513. https://doi.org/10.1016/S0076-6879(09)63029-9

    Article  CAS  PubMed  Google Scholar 

  7. Garfin DE (1990) One-dimensional gel electrophoresis. Methods Enzymol 182:425–441

    Article  CAS  PubMed  Google Scholar 

  8. Nowakowski AB, Wobig WJ, Petering DH (2014) Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics 6:1068–1078. https://doi.org/10.1039/c4mt00033a

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Zhou J, Dann GP, Shi T et al (2013) A simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics. Anal Chem 84:2862–2867. https://doi.org/10.1021/ac203394r

    Article  CAS  Google Scholar 

  11. Goldberg M, Expert-Bezancon N, Vuillard L et al (1996) Non-detergent sulphobetaines: a new class of molecules that facilitate in vitro protein renaturation. Fold Des 1(1):21–27 Current Biology Ltd ISSN 1359-0278

    Article  CAS  PubMed  Google Scholar 

  12. Sundaram RK, Balasubramaniyan N, Sundaram P (2012) Protein stains and applications. Methods Mol Biol 869:451–464. https://doi.org/10.1007/978-1-61779-821-4_39

    Article  CAS  PubMed  Google Scholar 

  13. Kurien BT, Scofield RH (2012) A brief review of other notable protein detection methods on acrylamide gels. Methods Mol Biol 869:617–620. https://doi.org/10.1007/978-1-61779-821-4_56

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Karlinsey JM (2012) Sample introduction techniques for microchip electrophoresis: a review. Anal Chim Acta 725:1–13. https://doi.org/10.1016/j.aca.2012.02.052

    Article  CAS  PubMed  Google Scholar 

  15. Amershamâ„¢ (2014) Automated Western blotting systems. Protein labeling and detection. Application note 29-1138-92 AB 09/2014

    Google Scholar 

  16. Amershamâ„¢ (2014) Analysis of therapeutic antibodies using Amershamâ„¢. WB system. Protein labeling and detection. Application note 29-1140-27

    Google Scholar 

  17. Amershamâ„¢ (2014) Quantitative fluorescence Western blot using Amershamâ„¢. WB system. Protein labeling and detection. Application note 29-1138-93

    Google Scholar 

  18. Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874

    Article  CAS  PubMed  Google Scholar 

  19. Engvall E, Jonsson K, Perlmann P (1971) Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 251:427–434

    Article  CAS  PubMed  Google Scholar 

  20. Koehler G, Milstein C (2005) Continuous cultures of fused cells secreting antibody of predefined specificity. J Immunol 174:2453–2465

    CAS  Google Scholar 

  21. Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51:2415–2418. https://doi.org/10.1373/clinchem.2005.051532

    Article  CAS  PubMed  Google Scholar 

  22. Aydin S (2015) A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 72:4–15. https://doi.org/10.1016/j.peptides.2015.04.012

    Article  CAS  PubMed  Google Scholar 

  23. Cheng HM (1996) Tween 20 selectively enhances naturally occurring anticardiolipin antibody binding in ELISA procedures. J Immunol Methods 191:87–88. https://doi.org/10.1016/0022-1759(96)00016-6

    Article  CAS  PubMed  Google Scholar 

  24. Cabral AR, Cabiedes J, Alarcón-Segovia D (1994) Tween 20 detaches cardiolipin from ELISA plates and makes anticardiolipin antibodies undetectable regardless of the presence of beta 2-glycoprotein-I. J Immunol Methods 175:107–114

    Article  CAS  PubMed  Google Scholar 

  25. Nielsen UB, Geierstanger BH (2004) Multiplexed sandwich assays in microarray format. J Immunol Methods 290:107–120. https://doi.org/10.1016/j.jim.2004.04.012

    Article  CAS  PubMed  Google Scholar 

  26. Zhang QY, Chen H, Lin Z, Lin JM (2012) Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein. J Pharm Anal 2:130–135. https://doi.org/10.1016/j.jpha.2011.10.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gao Z, Hou L, Xu M, Tang D (2015) Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein. Sci Rep 4:1–8. https://doi.org/10.1038/srep03966

    Article  CAS  Google Scholar 

  28. Larsson A, Holmdahl R (1990) A microELISA useful for determination of protein A-binding monoclonal antibodies. Hybridoma 9:289–294. https://doi.org/10.1089/hyb.1990.9.289

    Article  CAS  PubMed  Google Scholar 

  29. Maccani A, Landes N, Stadlmayr G et al (2014) Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol J 9:526–537. https://doi.org/10.1002/biot.201300305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu R, Lin Q, Sun Y et al (2009) Expression, purification, and characterization of hepatitis B virus surface antigens (HBsAg) in yeast Pichia pastoris. Appl Biochem Biotechnol 158:432–444. https://doi.org/10.1007/s12010-009-8527-x

    Article  CAS  PubMed  Google Scholar 

  31. Howard JW, Kay RG, Pleasance S, Creaser CS (2012) UHPLC for the separation of proteins and peptides. Bioanalysis 4:2971–2988. https://doi.org/10.4155/bio.12.283

    Article  CAS  PubMed  Google Scholar 

  32. Nováková L, Solichová D, Solich P (2006) Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel. J Sep Sci 29:2433–2443

    Article  PubMed  Google Scholar 

  33. Andrés A, Broeckhoven K, Desmet G (2015) Methods for the experimental characterization and analysis of the efficiency and speed of chromatographic columns: a step-by-step tutorial. Anal Chim Acta 894:20–34. https://doi.org/10.1016/j.aca.2015.08.030

    Article  CAS  PubMed  Google Scholar 

  34. Vehovec T, Obreza A (2010) Review of operating principle and applications of the charged aerosol detector. J Chromatogr A 1217:1549–1556. https://doi.org/10.1016/j.chroma.2010.01.007

    Article  CAS  PubMed  Google Scholar 

  35. Fekete S, Veuthey JL, Guillarme D (2015) Comparison of the most recent chromatographic approaches applied for fast and high resolution separations: theory and practice. J Chromatogr A 1408:1–14. https://doi.org/10.1016/j.chroma.2015.07.014

    Article  CAS  PubMed  Google Scholar 

  36. Gurramkonda C, Polez S, Skoko N et al (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Factories 9(31):1–11. https://doi.org/10.1186/1475-2859-9-31

    Article  CAS  Google Scholar 

  37. Polez S, Origi D, Zahariev S et al (2016) A simplified and efficient process for insulin production in Pichia pastoris. PLoS One 11:1–15. https://doi.org/10.1371/journal.pone.0167207

    Article  CAS  Google Scholar 

  38. Gurramkonda C, Adnan A, Gäbel T et al (2009) Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. Microb Cell Factories 8(13):1–8. https://doi.org/10.1186/1475-2859-8-13

    Article  CAS  Google Scholar 

  39. Heo J-H, Won HS, Kang HA et al (2002) Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha. Protein Expr Purif 24:117–122. https://doi.org/10.1006/prep.2001.1527

    Article  CAS  PubMed  Google Scholar 

  40. Ye J, Ly J, Watts K et al (2011) Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production. Biotechnol Prog 27:1744–1750. https://doi.org/10.1002/btpr.695

    Article  CAS  PubMed  Google Scholar 

  41. Applied Biosystems (2010) POROS A20 Analytical HPLC columns for the Quantitation of Monoclonal antibodies. Application Note 1-13

    Google Scholar 

  42. Moore JD, Perez-Pardo MA, Popplewell JF et al (2011) Chemical and biological characterisation of a sensor surface for bioprocess monitoring. Biosens Bioelectron 26:2940–2947. https://doi.org/10.1016/j.bios.2010.11.043

    Article  CAS  PubMed  Google Scholar 

  43. Petersen R (2017) Strategies using bio-layer interferometry biosensor technology for vaccine research and development. Biosensors 7(49):1–15. https://doi.org/10.3390/bios7040049

    Article  Google Scholar 

  44. Fortebio P (2009) MAb quantitation: protein A HPLC vs. protein A bio-layer interferometry. Application Note 15:1–21

    Google Scholar 

  45. Fortebio P (2009) High sensitivity detection of human IgG using protein A biosensors. Technical Note 15:1–9

    Google Scholar 

  46. Gilmore J, Islam M, Martinez-Duarte R (2016) Challenges in the use of compact disc-based centrifugal microfluidics for healthcare diagnostics at the extreme point of care. Micromachines 7:1–26. https://doi.org/10.3390/mi7040052

    Article  Google Scholar 

  47. Gyros (2015) Instruction For Use Gyrolabâ„¢ huIgG Kit, D0024808/C:1-10

    Google Scholar 

  48. Liedberg B, Nylander C, Lundström I (1995) Biosensing with surface plasmon resonance—how it all started. Biosens Bioelectron 10:i–ix

    Article  CAS  PubMed  Google Scholar 

  49. Karlsson R, Larsson A (2004) Affinity measurement using surface plasmon resonance. Methods Mol Biol 248:389–415

    CAS  PubMed  Google Scholar 

  50. Reinartz HW, Quinn JG, Zänker K, O’Kennedy R (1996) Bispecific multivalent antibody studied by real-time interaction analysis for the development of an antigen-inhibition enzyme-linked immunosorbent assay. Analyst 121:767–771

    Article  CAS  PubMed  Google Scholar 

  51. Mcdonnell JM (2001) Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition. Curr Opin Chem Biol 5:572–577

    Article  CAS  PubMed  Google Scholar 

  52. Nelson RW, Nedelkov D, Tubbs KA (2000) Biosensor chip mass spectrometry: a chip-based proteomics approach. Electrophoresis 21:1155–1163. https://doi.org/10.1002/(SICI)1522-2683(20000401)21:6<1155::AID-ELPS1155>3.0.CO;2-X

    Article  CAS  PubMed  Google Scholar 

  53. Hartmann-Petersen R, Gordon C (2005) Quantifying protein-protein interactions in the ubiquitin pathway by surface plasmon resonance. Methods Enzymol 399:164–177. https://doi.org/10.1016/S0076-6879(05)99011-3

    Article  CAS  PubMed  Google Scholar 

  54. Hellwig S, Emde F, Raven NPG et al (2001) Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. Biotechnol Bioeng 74:344–352. https://doi.org/10.1002/bit.1125

    Article  CAS  PubMed  Google Scholar 

  55. GE Healthcare Life Sciences (2008) Sensor Chip NTA. Instruction 22-0519-97:1–11

    Google Scholar 

  56. GE Healthcare Life Sciences (2011) Sensor Chip NTA and NTA Reagent Kit Data file 29-0079-27 AA:14

    Google Scholar 

  57. Namba Y, Usami M, Suzuki O (1999) Highly sensitive electrochemiluminescence immunoassay using the ruthenium chelate-labeled antibody bound on the magnetic micro beads. Anal Sci 15:1087–1093. https://doi.org/10.2116/analsci.15.1087

    Article  CAS  Google Scholar 

  58. Sanchez-Carbayo M, Espasa A, Chinchilla V et al (1999) New electrochemiluminescent immunoassay for the determination of CYFRA: analytical evaluation and clinical diagnostic performance in urine samples of patients with bladder cancer. Clin Chem 45:1944–1953

    CAS  PubMed  Google Scholar 

  59. Meso Scale Discovery (2012) MULTI-ARRAY® Assay System Human Insulin Kit 17099-v5-2012May:1-15

    Google Scholar 

  60. Buhlmann C, Preckel T, Chan S et al (2003) A new tool for routine testing of cellular protein expression: integration of cell staining and analysis of protein expression on a microfluidic chip-based system. J Biomol Techniques 14:119–127

    Google Scholar 

  61. Pandey S, Lu CM, Herold DA (2008) Measurement of microalbuminuria using protein chip electrophoresis. Am J Clin Pathol 129:432–438. https://doi.org/10.1309/4JU0XQH62D3YLTGK

    Article  CAS  PubMed  Google Scholar 

  62. Caliper Life Sciences (2009) Antibody analysis using Caliper’s LabChip GXII system. Application Note 400:1–4

    Google Scholar 

  63. Caliper Life Sciences (2009) Automated analysis of proteins using the LabChip 90 System. Application Note 100:1–4

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria

    Karola Vorauer-Uhl & Gabriele Lhota

Authors
  1. Karola Vorauer-Uhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriele Lhota
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karola Vorauer-Uhl .

Editor information

Editors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Brigitte Gasser

  2. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Diethard Mattanovich

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Vorauer-Uhl, K., Lhota, G. (2019). Quantification of Recombinant Products in Yeast. In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9024-5_20

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9023-8

  • Online ISBN: 978-1-4939-9024-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation