TAP isolation of Ras protein complexes from mammalian cells

Transfection
Transfect 5x10⁸ NIH 3T3 cells with TAP-Ras fusion constructs using Lipofectamine (Invitrogen).

Perform all steps in a laminar flow hood, with gloves pulled over clamped disposable sleeves. Use only unused pipets, pipet tips, dishes and disposable columns and clean all pipettors and equipment thoroughly with 70% ethanol before use.

Cell lysis
Wash cells 3x with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Remove last traces of PBS.  Freeze dishes at -80°C overnight.
Lyse cells:
Scrape cells into 1 ml lysis buffer* + 1 mM EGTA.  Keep on ice 30 min.
Remove insoluble material by centrifugation at 14000xg for 10 min at 4°C.

Binding to Ni++ beads
Transfer 400 ul 1:1 Ni++-charged sepharose beads to a 10ml Bio-Rad disposable filtered column, wash with 5 ml lysis buffer + 40 mM imidazole.
Cap bottom of column, add cell lysate, cap the top, and incubate 1 hr at 4°C in constant rotation.
Remove cap and drain column.
Wash beads with 30 ml lysis buffer + 40 mM imidazole.

Elution from Ni++ beads with imidazole
Elute in 5 ml lysis buffer + 400 mM imidazole + 2 mM CaCl2.

Binding to calmodulin resin
In a fresh column, add 100 ul 1:1 calmodulin beads.  Wash with 5 ml lysis buffer + 400 mM imidazole + 2 mM CaCl2.
Cap columns.  Add Ni++ eluates to columns and cover.  Incubate 1 hr 4°C in constant rotation.
Wash columns with 5 ml lysis buffer + 400 mM imidazole + 2 mM CaCl2.
Wash with 5 ml lysis buffer + 400 mM imidazole + 2 mM CaCl2 with no detergent.

Elution from calmodulin columns
Elute columns in 200 ul lysis buffer + 4 mM EGTA with no detergent.
Send snap-frozen eluates for LC/MS analysis.

* Lysis buffer: 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2 mM MgCl2, 0.5% NP-40, plus freshly added protease inhibitor cocktail (Roche) and 10 uM GTP.

N-terminal TAP-tagged GTPase constructs

nTAP: pEF4 expression construct containing the N-terminal TAP tag. Coding sequences were cloned in frame for generation of a fusion construct using BamH1 (5’).

nTAP-RRas(G38V)**

nTAP-RRas(T43N)**

nTAP-HRas(G12V)**

nTAP-HRas(T17N)

nTAP-Rap1a(G12V)

nTAP-Rap1a(T17N)

nTAP-KRas(G12V)

nTAP-KRas(T17N)

 

 

These constructs can obtained from Lawrence Goldfinger (lgoldfinger@ucsd.edu) or Mark Ginsberg (mhginsberg@ucsd.edu).

Confirmation that TAP-Ras fusion constructs are functional in vivo.

To confirm that full-length Ras constructs maintain Ras functions in vivo in the presence of a TAP fusion, we analyzed the ability of TAP fusions of distinct Ras isotypes to modulate integrin activation, as previously observed (Oertli et al., 2000). Expression of activated H-Ras(G12V) suppresses integrin activation in CHO cells bearing chimeric integrins aIIba6b3b1, which are constitutively active, as measured by cell labeling with the ligand-mimetic antibody, PAC1, a generous gift of Dr. S. Shattil (Shattil et al., 1985; Hughes et al., 1997).  Activated R-Ras reverses integrin suppression, and an additional mutation in the effector loop (G38VD64A) does not disrupt this function of R-Ras. However, an alternate mutation of the same residue (G38VD64E) blocks the ability of R-Ras to reverse suppression of integrin activation by activated H-Ras (Oertli et al., 2000).  As shown below, TAP fusion to H-Ras and R-Ras did not alter the abilities of these GTPases to modulate integrin activation in cells.

** These constructs have been confirmed for functionality by the integrin activation assay.

References

Hughes,P.E., Renshaw,M.W., Pfaff,M., Forsyth,J., Keivens,V.M., Schwartz,M.A., and Ginsberg,M.H. (1997). Suppression of integrin activation: A novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 88, 521-530. PubMed
Oertli,B., Han,J., Marte,B.M., Sethi,T., Downward,J., Ginsberg,M., and Hughes,P.E. (2000). The effector loop and prenylation site of R-Ras are involved in the regulation of integrin function [In Process Citation]. Oncogene 19, 4961-4969. PubMed
Shattil,S.J., Brass,L.F., Bennett,J.S., and Pandhi,P. (1985). Biochemical and Functional Consequences of Dissociation of the Platelet Membrane Glycoprotein IIb-IIIa Complex. Blood 66, 92-98. PubMed

 

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