pH responsive granulocyte colony-stimulating factor variants with implications for treating Alzheimer's disease and other central nervous system disorders

Pete Heinzelman*, Jennifer A. Schoborg, Michael C. Jewett

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Systemic injection of granulocyte colony-stimulating factor (G-CSF) has yielded encouraging results in treating Alzheimer's Disease (AD) and other central nervous system (CNS) disorders. Making G-CSF a viable AD therapeutic will, however, require increasing G-CSF's ability to stimulate neurons within the brain. This objective could be realized by increasing transcytosis of G-CSF across the blood brain barrier (BBB). An established correlation between G-CSF receptor (G-CSFR) binding pH responsiveness and increased recycling of G-CSF to the cell exterior after endocytosis motivated development of G-CSF variants with highly pH responsive G-CSFR binding affinities. These variants will be used in future validation of our hypothesis that increased BBB transcytosis can enhance G-CSF therapeutic efficacy. Flow cytometric screening of a yeast-displayed library in which G-CSF/G-CSFR interface residues were mutated to histidine yielded a G-CSF triple His mutant (L109H/D110H/Q120H) with highly pH responsive binding affinity. This variant's KD, measured by surface plasmon resonance (SPR), increases ∼20-fold as pH decreases from 7.4 to below histidine's pKa of ∼6.0; an increase 2-fold greater than for previously reported G-CSF His mutants. Cell-free protein synthesis (CFPS) enabled expression and purification of soluble, bioactive G-CSF triple His variant protein, an outcome inaccessible via Escherichia coli inclusion body refolding. This purification and bioactivity validation will enable future identification of correlations between pH responsiveness and transcytosis in BBB cell culture model and animal experiments. Furthermore, the library screening and CFPS methods employed here could be applied to developing other pH responsive hematopoietic or neurotrophic factors for treating CNS disorders.

Original languageEnglish (US)
Pages (from-to)481-489
Number of pages9
JournalProtein Engineering, Design and Selection
Volume28
Issue number10
DOIs
StatePublished - Oct 2015

Keywords

  • Alzheimer's disease
  • blood brain barrier
  • cell-free protein synthesis
  • granulocyte colony-stimulating factor
  • yeast surface display

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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