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

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

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 date2017
DOIs
Publication statusPublished - 2018
MoE publication typeA1 Journal article-refereed

Keywords

  • antibody
  • hydrophobin
  • Nicotiana benthamiana
  • Protein A
  • puri?cation
  • tobacco BY-2
  • suspension cells

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