Improved expression of recombinant plant-made hEGF

David Rhys Thomas, Amanda Maree Walmsley

Research output: Contribution to journalArticleResearchpeer-review

10 Citations (Scopus)

Abstract

Key message: The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.

Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

Original languageEnglish
Pages (from-to)1801-1814
Number of pages14
JournalPlant Cell Reports
Volume33
Issue number11
DOIs
Publication statusPublished - 2014

Keywords

  • Codon optimisation
  • Human epidermal growth factor
  • Nicotiana benthamiana
  • Sub-cellular targeting
  • Transient expression
  • Unfolded protein response

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