Elsevier

Journal of Biotechnology

Volume 219, 10 February 2016, Pages 24-27
Journal of Biotechnology

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

https://doi.org/10.1016/j.jbiotec.2015.12.020Get rights and content
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open access

Highlights

  • Fusion protein constructs of horseradish peroxidase and protein G were produced.

  • The setup of the fusion constructs strongly affected the yields.

  • The process yielded a functional protein without the need for chemical conjugation.

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.

Abbreviations

ABTS
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt
BSM
basal salts medium
BMGY
buffered minimal glycerol yeast medium
BSA
bovine serum albumin
Ca-TBS
calcium-supplemented tris–buffered saline
DCW
dry cell weight
ELISA
enzyme-linked immunosorbent assays
HRP
horseradish peroxidase
IgG
immunoglobulin G
PTM1
trace element solution
SpG
streptococcal protein G
TMB
3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate
YE
Bacto™ yeast extract
YNB
difco™ yeast nitrogen base w/o amino acids

Keywords

Antibody
Diagnostics
Fusion protein
Peroxidase
Protein G

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