PMID- 25015885
OWN - NLM
STAT- MEDLINE
DCOM- 20160208
LR  - 20211021
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Print)
IS  - 0099-2240 (Linking)
VI  - 80
IP  - 18
DP  - 2014 Sep
TI  - Directed evolution of brain-derived neurotrophic factor for improved folding and 
      expression in Saccharomyces cerevisiae.
PG  - 5732-42
LID - 10.1128/AEM.01466-14 [doi]
AB  - Brain-derived neurotrophic factor (BDNF) plays an important role in nervous 
      system function and has therapeutic potential. Microbial production of BDNF has 
      resulted in a low-fidelity protein product, often in the form of large, insoluble 
      aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, 
      employing Saccharomyces cerevisiae display and secretion systems, it was found 
      that BDNF was poorly expressed and partially inactive on the yeast surface and 
      that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. 
      Thus, for the purpose of increasing the compatibility of yeast as an expression 
      host for BDNF, directed-evolution approaches were employed to improve BDNF 
      folding and expression levels. Yeast surface display was combined with two rounds 
      of directed evolution employing random mutagenesis and shuffling to identify BDNF 
      mutants that had 5-fold improvements in expression, 4-fold increases in specific 
      TrkB binding activity, and restored p75 binding activity, both as displayed 
      proteins and as secreted proteins. Secreted BDNF mutants were found largely in 
      the form of soluble homodimers that could stimulate TrkB phosphorylation in 
      transfected PC12 cells. Site-directed mutagenesis studies indicated that a 
      particularly important mutational class involved the introduction of cysteines 
      proximal to the native cysteines that participate in the BDNF cysteine knot 
      architecture. Taken together, these findings show that yeast is now a viable 
      alternative for both the production and the engineering of BDNF.
CI  - Copyright (c) 2014, American Society for Microbiology. All Rights Reserved.
FAU - Burns, Michael L
AU  - Burns ML
AD  - Department of Chemical and Biological Engineering, University of 
      Wisconsin-Madison, Madison, Wisconsin, USA.
FAU - Malott, Thomas M
AU  - Malott TM
AD  - Department of Chemical and Biological Engineering, University of 
      Wisconsin-Madison, Madison, Wisconsin, USA.
FAU - Metcalf, Kevin J
AU  - Metcalf KJ
AD  - Department of Chemical and Biological Engineering, University of 
      Wisconsin-Madison, Madison, Wisconsin, USA.
FAU - Hackel, Benjamin J
AU  - Hackel BJ
AD  - Department of Chemical Engineering and Materials Science, University of 
      Minnesota, Minneapolis, Minnesota, USA.
FAU - Chan, Jonah R
AU  - Chan JR
AD  - Department of Neurology, Program in Neuroscience, University of California, San 
      Francisco, San Francisco, California, USA.
FAU - Shusta, Eric V
AU  - Shusta EV
AD  - Department of Chemical and Biological Engineering, University of 
      Wisconsin-Madison, Madison, Wisconsin, USA shusta@engr.wisc.edu.
LA  - eng
GR  - P30 CA014520/CA/NCI NIH HHS/United States
GR  - T32 DK007007/DK/NIDDK NIH HHS/United States
GR  - T90DK07007/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20140711
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Brain-Derived Neurotrophic Factor)
RN  - 0 (Membrane Glycoproteins)
RN  - EC 2.7.10.1 (Protein-Tyrosine Kinases)
RN  - EC 2.7.10.1 (Receptor, trkB)
RN  - EC 2.7.10.1 (tropomyosin-related kinase-B, human)
SB  - IM
MH  - Brain-Derived Neurotrophic Factor/*genetics/*metabolism
MH  - Cell Surface Display Techniques
MH  - DNA Shuffling
MH  - *Directed Molecular Evolution
MH  - Gene Expression
MH  - Membrane Glycoproteins/metabolism
MH  - Mutagenesis, Site-Directed
MH  - Protein Binding
MH  - *Protein Folding
MH  - Protein-Tyrosine Kinases/metabolism
MH  - Receptor, trkB
MH  - Saccharomyces cerevisiae/*genetics
PMC - PMC4178591
EDAT- 2014/07/13 06:00
MHDA- 2016/02/09 06:00
PMCR- 2015/03/01
CRDT- 2014/07/13 06:00
PHST- 2014/07/13 06:00 [entrez]
PHST- 2014/07/13 06:00 [pubmed]
PHST- 2016/02/09 06:00 [medline]
PHST- 2015/03/01 00:00 [pmc-release]
AID - AEM.01466-14 [pii]
AID - 01466-14 [pii]
AID - 10.1128/AEM.01466-14 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2014 Sep;80(18):5732-42. doi: 10.1128/AEM.01466-14. Epub 
      2014 Jul 11.