Proteome profiling and vector yield optimization in a recombinant adeno-associated virus-producing yeast model.
Animals
Cell Line
Dependovirus
/ genetics
Gene Expression Profiling
Heat-Shock Proteins
/ metabolism
Mass Spectrometry
Oxidative Stress
/ genetics
Plasmids
/ genetics
Proteome
/ metabolism
Reactive Oxygen Species
/ metabolism
Saccharomyces cerevisiae
/ genetics
Sf9 Cells
Spodoptera
Unfolded Protein Response
/ genetics
Viral Proteins
/ biosynthesis
Saccharomyces
adeno-associated virus
gene therapy
mass spectrometry
Journal
MicrobiologyOpen
ISSN: 2045-8827
Titre abrégé: Microbiologyopen
Pays: England
ID NLM: 101588314
Informations de publication
Date de publication:
12 2020
12 2020
Historique:
received:
31
07
2020
revised:
13
10
2020
accepted:
14
10
2020
pubmed:
10
11
2020
medline:
17
8
2021
entrez:
9
11
2020
Statut:
ppublish
Résumé
Recent studies on recombinant adeno-associated viral (rAAV) vector production demonstrated the generation of infectious viral particles in Saccharomyces cerevisiae. Proof-of-concept results showed low vector yields that correlated with low AAV DNA encapsidation rates. In an attempt to understand the host cell response to rAAV production, we profiled proteomic changes throughout the fermentation process by mass spectrometry. By comparing an rAAV-producing yeast strain with a respective non-producer control, we identified a subset of yeast host proteins with significantly different expression patterns during the rAAV induction period. Gene ontology enrichment and network interaction analyses identified changes in expression patterns associated mainly with protein folding, as well as amino acid metabolism, gluconeogenesis, and stress response. Specific fold change patterns of heat shock proteins and other stress protein markers suggested the occurrence of a cytosolic unfolded protein response during rAAV protein expression. Also, a correlative increase in proteins involved in response to oxidative stress suggested cellular activities to ameliorate the effects of reactive oxygen species or other oxidants. We tested the functional relevance of the identified host proteins by overexpressing selected protein leads using low- and high-copy number plasmids. Increased vector yields up to threefold were observed in clones where proteins SSA1, SSE1, SSE2, CCP1, GTT1, and RVB2 were overexpressed. Recombinant expression of SSA1 and YDJ insect homologues (HSP40 and HSC70, respectively) in Sf9 cells led to a volumetric vector yield increase of 50% relative to control, which validated the importance of chaperone proteins in rAAV-producing systems. Overall, these results highlight the utility of proteomic-based tools for the understanding and optimization of rAAV-producing recombinant strains.
Identifiants
pubmed: 33166081
doi: 10.1002/mbo3.1136
pmc: PMC7755776
doi:
Substances chimiques
Heat-Shock Proteins
0
Proteome
0
Reactive Oxygen Species
0
Viral Proteins
0
Banques de données
figshare
['10.6084/m9.figshare.13040591.v1']
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1136Informations de copyright
© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
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