Recombinant production of a peroxidase-protein G fusion protein in Pichia pastoris

J Biotechnol. 2016 Feb 10;219:24-7. doi: 10.1016/j.jbiotec.2015.12.020. Epub 2015 Dec 18.

Abstract

Streptococcal protein G (SpG) binds immunoglobulin G from a broad range of mammalian species with high affinity. Chemical conjugations of SpG to the reporter enzyme horseradish peroxidase (HRP) are commonly used in immunohistochemical applications. However, commercial HRP preparations are typically isolated from horseradish roots as varying mixtures of HRP isoenzymes with different biochemical properties, and chemical conjugation procedures lead to heterogeneous HRP-SpG preparations, partially including inactivated enzyme. A recombinant process allows the production of a well-defined HRP isoenzyme fused to SpG at constant 1:1 stoichiometry in a single step without the need for laborious chemical conjugation. By using state-of-the-art biotechnological tools, we produced a recombinant HRP-SpG fusion protein in Pichia pastoris in bioreactor cultivations. Purified HRP-SpG was tested successfully for functional binding of antibodies from different mammalian serums. Recombinant production of this novel well-defined fusion protein follows quality-by-design principles and facilitates the production of more reliable and cost-effective diagnostic kits.

Keywords: Antibody; Diagnostics; Fusion protein; Peroxidase; Protein G.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Batch Cell Culture Techniques
  • Bioreactors
  • Fungal Proteins / genetics
  • Fungal Proteins / immunology
  • Fungal Proteins / isolation & purification
  • Horseradish Peroxidase / genetics
  • Horseradish Peroxidase / metabolism*
  • Isoenzymes / genetics
  • Isoenzymes / immunology
  • Isoenzymes / isolation & purification
  • Pichia / genetics*
  • Pichia / metabolism
  • Protein Engineering
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / isolation & purification*

Substances

  • Antibodies
  • Bacterial Proteins
  • Fungal Proteins
  • IgG Fc-binding protein, Streptococcus
  • Isoenzymes
  • Recombinant Fusion Proteins
  • Horseradish Peroxidase