Abstract

In TR&D 1, we develop approaches to make complex changes to cells, and we generate biosensors to report on the effects of some of these changes. In the first aim, we establish strategies to rapidly and combinatorially up- or down-regulate a set of genes in a common pathway in order to improve the expression of a desired protein, and optimize these approaches using a vaccine protein that is commercially made in yeast. In the second aim, we devise new selections for use in chemostats and turbidostats to identify the effects of small changes in proteins, in promoters and in untranslated regions of mRNAs. Further, we modify the turbidostat platform to enable ramped selections, allowing feedback on temperature or drug concentration to optimize selections. In the third aim, we design new proteins that bind to a protein-protein interface and disrupt an interaction. These inhibitory proteins will allow us to assess the roles of individual members of large protein families that are difficult to analyze. Finally, in the fourth aim, we generate new biosensors to detect the levels of a small molecule and of a protein produced in yeast.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Biotechnology Resource Grants (P41)
Project #
2P41GM103533-21
Application #
9208071
Study Section
Special Emphasis Panel (ZRG1)
Project Start
1997-09-30
Project End
2022-03-31
Budget Start
2017-01-01
Budget End
2017-12-31
Support Year
21
Fiscal Year
2017
Total Cost
Indirect Cost

  Institution

Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Starita, Lea M; Islam, Muhtadi M; Banerjee, Tapahsama et al. (2018) A Multiplex Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function. Am J Hum Genet 103:498-508
Wang, Zheng; Wu, Catherine; Aslanian, Aaron et al. (2018) Defective RNA polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 pathway. Elife 7:
Andeen, Nicole K; Yang, Han-Yin; Dai, Dao-Fu et al. (2018) DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN. J Am Soc Nephrol 29:231-239
Helgeson, Luke A; Zelter, Alex; Riffle, Michael et al. (2018) Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments. Proc Natl Acad Sci U S A 115:2740-2745
Fong, Kimberly K; Zelter, Alex; Graczyk, Beth et al. (2018) Novel phosphorylation states of the yeast spindle pole body. Biol Open 7:
González, Delfina P; Lamb, Helen V; Partida, Diana et al. (2018) CBD-1 organizes two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans. Dev Biol 442:288-300
Basisty, Nathan B; Liu, Yuxin; Reynolds, Jason et al. (2018) Stable Isotope Labeling Reveals Novel Insights Into Ubiquitin-Mediated Protein Aggregation With Age, Calorie Restriction, and Rapamycin Treatment. J Gerontol A Biol Sci Med Sci 73:561-570
Brandsen, Benjamin M; Mattheisen, Jordan M; Noel, Teia et al. (2018) A Biosensor Strategy for E. coli Based on Ligand-Dependent Stabilization. ACS Synth Biol 7:1990-1999
Ma, Yuanhui; Yates 3rd, John R (2018) Proteomics and pulse azidohomoalanine labeling of newly synthesized proteins: what are the potential applications? Expert Rev Proteomics 15:545-554
Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E et al. (2018) A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack. MBio 9:

Showing the most recent 10 out of 372 publications