The authors have no conflict of interest.
Cis-suppression to arrest protein aggregation in mammalian cells
Article first published online: 18 OCT 2013
© 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
How to Cite
Gregoire, S., Zhang, S., Costanzo, J., Wilson, K., Fernandez, E. J. and Kwon, I. (2013), Cis-suppression to arrest protein aggregation in mammalian cells. Biotechnol. Bioeng.. doi: 10.1002/bit.25119
- Article first published online: 18 OCT 2013
- Accepted manuscript online: 23 SEP 2013 02:22AM EST
- Manuscript Accepted: 9 SEP 2013
- Manuscript Revised: 18 AUG 2013
- Manuscript Received: 22 MAY 2013
- National Institutes of Health. Grant Number: R21NS069946
- KSEA Young Investigator
- Korea CCS R&D Center (KCRC). Grant Number: 2013M1A8A1038187
- NSF PAGES
- protein aggregation;
- protein engineering;
- mammalian cells;
- superoxide dismutase;
Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1A4V) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1A4V as a key residue responsible for SOD1A4V conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays. Biotechnol. Bioeng. 2013;xxx: xxx–xxx. © 2013 Wiley Periodicals, Inc.