RAS Tools and Resources

DNA Reagents

Complete Set of RAS Pathway Genes

Access: Visit Addgene for the RAS Pathway 2.0 Clone Collection

This collection of 180 genes comprises the core of the RAS pathway. After crowdsourcing the pathway map, the RAS Initiative constructed entry clones of each gene representing the dominant transcript across more than 30 human cancers.

All 180 genes—including stop and no-stop versions—are available. You may use the Gateway Cloning Platform (Life Technologies) to easily convert these to expression clones for a variety of experimental purposes.

KRAS Entry Clone Collection

Access: Visit Addgene for the RAS Mutant Clone Collection

This collection of plasmids contains the full-length sequences of verified entry clones for KRAS4b, KRAS4a, HRAS, and NRAS, along with a variety of oncogenic mutants of the isoforms.

These can be utilized to generate expression clones in standard Gateway compatible destination vectors.

RAS Superfamily Clone Collection

Access: Coming soon to Addgene

This collection of plasmids contains the full-length sequence of verified entry clones of all RAS Superfamily members: KRAS4a, KRAS4b, HRAS, NRAS, RIT1, RIT12, DIRAS1, DIRAS2, DIRAS3, ERAS, GEM, MRAS, NKIRAS1, NKIRAS2, RALA, RALB, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, RASD1, RASD2, RRAS, RRAS2, RASL10A, RASL10B, RASL11A, RASL11B, RASL12, RHEB, RHEBL1, REM1, REM2, RERG, RERGL, and RRAD.

The RAS Initative generated this collection to serve as a follow-on to its initial KRAS Entry Clone Collection to study the larger family of RAS-related proteins.

DNA Constructs for RAS Protein Production

Access: Visit Addgene for the Dominic Esposito Lab Materials or email Dominic Esposito (dom.esposito@nih.gov)

This collection contains more than 300 expression constructs to aid in the production of RAS and RAS-related proteins for biochemical assays and structural biology. It also includes 60 wild-type and mutant RAS genes as Gateway entry clones that are fully sequenced and have the same context to enable optimal correlation of phenotype with genotype.

Many of these constructs are available from the Addgene repository; others can be obtained by emailing Dominic Esposito directly.

Assay Reagents

Access: Email Dominic Esposito (dom.esposito@nih.gov)

The RAS Initiative offers a variety of materials for the deployment of biochemical, biophysical, and cell-based assays for drug discovery:

• DNA constructs: Available for the production of tagged proteins for biochemical assays including SPR (surface plasmon resonance), HTRF (homogeneous time-resolved fluorescence), and FRET (Forster resonance energy transfer) using Avi, GST, xFP, or Halotag fusions to RAS, RAF, and other RAS effectors.
• BRET (bioluminescence resonance energy transfer) assay clones: Available using Halotag and NanoLuc fusions to a variety of partner proteins including RAS/RAF, RAS/RALGDS, RAS/PIK3CA, SHOC2/MRAS, NF1/SPRED1, RAF/MEK, and RAS/RAS.
• Assay protocols and parameters: Available to enable researchers to quickly utilize these qualified assays for their screening needs.

Cell Line Reagents

RAS-dependent MEF (Mouse Embryonic Fibroblast) Cell Lines

Access: Email Billy Burgan (william.burgan2@nih.gov)

This collection of cell lines was developed from the original HRAS/NRAS knockout cell lines from the laboratory of Mariano Barbacid by removal of endogenous KRAS and replacement with a variety of mutant KRAS alleles.

All MEF cell lines undergo a variety of quality control measures:

1. Verification that endogenous KRAS has been removed
2. Integration analysis assays to identify the number and location of transgene insertions within the genome
3. Validation of final clones by exome sequencing to ensure that no additional mutations are present which could impact findings (such as mutations in other RAS pathway genes, or regulators like p53)
4. Testing of all cell lines for mycoplasma contamination
5. Authentication by short tandem repeat sequencing to further ensure the quality of reagents prior to distribution

Protein Production Tools

Production of Fully Processed KRAS-FMe Proteins

Access: Email Bill Gillette (gillettew@mail.nih.gov)

The RAS Initiative offers reagents for the high-yield production of fully processed KRAS proteins, including specialized insect cell lines, baculoviruses, and a variety of RAS mutant expression clones.

A major goal of the early part of the RAS Initiative was to develop appropriate reagents to examine the interaction of KRAS with membranes. To do this, it was necessary to have KRAS in its properly processed form consisting of a properly prenylated and methylated C-terminal region. Historically, this protein (called KRAS-FMe) could only be produced at low yield and purity. The RAS Initiative developed a process to improve this production by 50-fold using an engineered baculovirus system.

Chaperones for Improved Production of Proteins and Protein Complexes

Access: Email Dominic Esposito (dom.esposito@nih.gov)

The RAS Initiative offers chaperone families and generated research materials that can improve the production of a variety of proteins related to RAS research and aid in the production of proteins and protein complexes, including RAF kinases and the SHOC2/MRAS/PPP1CA phosphatase.

The RAS Initiative discovered that several complicated proteins and protein complexes often needed the assistance of folding chaperones for in vitro production. Now, we distribute specialized baculoviruses and DNA constructs containing these chaperones because they may be useful when working on related proteins.

Improved Insect Cell Protein Purification Technologies

Access: Email Dominic Esposito (dom.esposito@nih.gov)

The RAS Initiative offers the novel cell line Tni-FNL. This line can produce higher yields of protein than commercially available strains, as well as new baculovirus genomes with higher stability and increased protein production yields.