title: Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.
abstract: Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Burns
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Malott
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Metcalf
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Arthya
authors.initials: A
authors.lastname: Puguh
authors.affiliation: Department of Neurology, Program in Neuroscience, University of California, San Francisco, San Francisco, California, USA.
authors.forename: Jonah R
authors.initials: JR
authors.lastname: Chan
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA. shusta@engr.wisc.edu.
authors.forename: Eric V
authors.initials: EV
authors.lastname: Shusta
mesh_terms: D019208:Brain-Derived Neurotrophic Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020813:Receptor, trkB; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D008562:Membrane Glycoproteins; C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; C579455:BDNF protein, human; D011505:Protein-Tyrosine Kinases; D020813:Receptor, trkB; C000591259:tropomyosin-related kinase-B, human
keywords: Brain-derived neurotrophic factor; Directed evolution; Neurotrophin; Pro-region; Yeast surface display