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RAS Initiative Reagents Research

Triptych of a double helix, RAS mutations, and a crystal structure

Reagents developed by the RAS Reagents Group

The RAS Reagents Research Team supplies nucleic acids, cell lines, and protein reagents for the RAS Initiative. We support the development of structural studies, drug screens, imaging experiments, and cell biology and biochemistry projects. Many of our reagents are available to the community as RAS Reference Reagents.

Progress

We have expressed and purified fully modified KRAS protein. This form of KRAS is required for signaling inside cells. Significant quantities have not been available until now. View the publication.

We have provided insect cells and engineered baculovirus to researchers for producing their own fully modified KRAS protein. The reagents and protocols developed by FNLCR scientists are now available for distribution to the community.

Our team has been able to share 360 RAS pathway genes with the research community through Addgene. This is the only source for the entire set of RAS pathway genes. Importantly the isoform of each gene was chosen to match the forms most commonly found in human cancers.

The work of our group has led to the construction of >200 expression plasmids for protein purification, structural biology, cell line construction, and single molecule imaging studies. These are for use within the FNLCR, but will be made available as their utility is demonstrated.

Projects

  • Structural biology
  • Single molecule imaging
  • In vitro and in vivo drug screens
  • Screens for vulnerabilities in cancer cell lines

Tools

  • Gateway and Gateway Multisite cloning
  • Libraries of promoters, fluorescent proteins, purification tags, labeling tags
  • Bacterial, mammalian, insect, and viral vectors
  • RAS-less mouse embryonic fibroblasts

The RAS Initiative Reagents Research Team

Dominic Esposito headshot

Dr. Dom Esposito, RAS Reagents Research Team Lead

Team Lead and Contact

Dr. Dom Esposito
espositod@mail.nih.gov
301-846-7376

Publications

  • Snead K, Wall V, Ambrose H, Esposito D, Drew M. Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinant leucine-rich repeat proteins and protein complexes. Protein Expression and Purification. 2022;193:106061. doi:10.1016/j.pep.2022.106061 [PubMed Abstract]
  • Grose C, Putman Z, Esposito D. A review of alternative promoters for optimal recombinant protein expression in baculovirus-infected insect cells. Protein Expression and Purification. 2021;186:105924. doi:10.1016/j.pep.2021.105924 [PubMed Abstract]
  • Klumpp-Thomas C, Kalish H, Drew M, et al. Standardization of ELISA protocols for serosurveys of the SARS-COV-2 pandemic using clinical and at-home blood sampling. Nature Communications. 2021;12(1). doi:10.1038/s41467-020-20383-x [PubMed Abstract]
  • Pijlman GP, Grose C, Hick TA, et al. Relocation of the ATTTN7 transgene insertion site in Bacmid DNA enhances baculovirus genome stability and recombinant protein expression in insect cells. Viruses. 2020;12(12):1448. doi:10.3390/v12121448 [PubMed Abstract]
  • Gillette W, Frank P, Perkins S, Drew M, Grose C, Esposito D. Production of farnesylated and methylated proteins in an engineered insect cell system. Methods in Molecular Biology. 2019:259-277. doi:10.1007/978-1-4939-9532-5_20 [PubMed Abstract]
  • Esposito D, Stephen AG, Turbyville TJ, Holderfield M. New weapons to penetrate the armor: Novel reagents and assays developed at the NCI RAS initiative to enable discovery of Ras Therapeutics. Seminars in Cancer Biology. 2019;54:174-182. doi:10.1016/j.semcancer.2018.02.006 [PubMed Abstract]
  • Sherekar M, Han S-W, Ghirlando R, et al. Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer. Journal of Biological Chemistry. 2019;295(4):1105-1119. doi:10.1074/jbc.ra119.010934 [PubMed Abstract]
  • Procter L, Grose C, Esposito D. Production of authentic geranylgeranylated kras4b using an engineered Baculovirus System. Protein Expression and Purification. 2018;151:99-105. doi:10.1016/j.pep.2018.06.012 [PubMed Abstract]
  • Taylor T, Denson J-P, Esposito D. Optimizing expression and solubility of proteins in E. coli using modified media and induction parameters. Methods in Molecular Biology. 2017:65-82. doi:10.1007/978-1-4939-6887-9_5 [PubMed Abstract]
  • Mehalko JL, Esposito D. Engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells. Journal of Biotechnology. 2016;238:1-8. doi:10.1016/j.jbiotec.2016.09.002 [PubMed Abstract]
  • Gillette WK, Esposito D, Abreu Blanco M, et al. Farnesylated and methylated KRAS4b: High yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions. Scientific Reports. 2015;5(1). doi:10.1038/srep15916 [PubMed Abstract]
  • Wall VE, Garvey LA, Mehalko JL, Procter LV, Esposito D. Combinatorial Assembly of clone libraries using site-specific recombination. DNA Cloning and Assembly Methods. 2013:193-208. doi:10.1007/978-1-62703-764-8_14 [PubMed Abstract]
  • Updated:
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