(505d) Characterization of Pro-Regions in Mammalian Growth Factors Using Deep Sequencing and Yeast Surface Display

Growth factors comprise a large family of proteins that play a critical role in proliferation and differentiation, function as a cellular growth stimulator and inhibitors, and promote survival of cells. These proteins are associated with several diseases including neurodegenerative disorders, among others. These growth factors represent important targets for the development of potential therapeutic candidates. As one example, Brain-derived Neurotrophic Factor (BDNF) is a candidate to promote the regeneration of the lost or damage cells in combination with progenitor cells after a spinal cord injury¹. However, a major limitation is mammalian growth factor expression in active form in high titers. In their native context, certain growth factors are expressed as a pro-protein in which the pro-peptide may act as a molecular chaperone of the mature protein to guide correct folding and processing through the secretory pathway. In the attempt to achieve high titers, this pro-region has been engineered to enhance the folding and secretion of human BDNF for yeast expression². In this talk, we will present the improvement of the active titers of a particular growth factor using comprehensive site saturation mutagenesis (SSM) libraries of the pro-region coupled to yeast surface display and deep mutational scanning. These SSM libraries were also sorted against two different anti-growth factor monoclonal antibodies (mAbs) that are conformationally selective. We evaluated the function of 3,296 mutants and found over a 10-fold improvement in binding and folding activities after two round of sorting for approximately 86% of all possible single nonsynonymous mutations. Mutations that increased proper growth factor folding largely overlapped between the two mAbs, showing the reproducibility of the deep mutational scanning method. Distinct features of the pro-region that enable high yeast secretion will be discussed, as will downstream applications like conformational epitope mapping that are enabled by the high active surface expression of the growth factor.

1. Sahni, V. & Kessler, J. A. Stem cell therapies for spinal cord injury. Nat. Rev. Neurol. 6, 363–372 (2010).

2. Burns, M. L. et al. Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor. Biotechnol. J. 11, 425–436 (2016).