In-solution antibody harvesting with a plant-produced hydrophobin-Protein A fusion

Katri Kurppa, Lauri J. Reuter, Anneli Ritala, Markus B. Linder, Jussi J. Joensuu*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

11 Citations (Scopus)
194 Downloads (Pure)

Abstract

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.

Original languageEnglish
Pages (from-to)404-414
Number of pages11
JournalPLANT BIOTECHNOLOGY JOURNAL
Volume16
Issue number2
Early online date1 Aug 2017
DOIs
Publication statusPublished - Feb 2018
MoE publication typeA1 Journal article-refereed

Keywords

  • Nicotiana benthamiana
  • Antibody
  • Hydrophobin
  • Protein A
  • Purification
  • Tobacco BY-2 suspension cells

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