Use of the insect/baculovirus expression system to produce lipid-modified ras proteins for electrospray mass spectrometric studies

Abstract

We describe methods for the production of lipid-modified ras proteins using the insect/baculovirus expression system. Protocols for labeling these proteins with precursors of these modifications, e.g., [ methyl - 3 H]methionine, [ 3 H]mevalonic acid, and [ 3 H]palmitate, are given. In addition, we detail the purification of the proteins using ion-exchange chromatography and immunoaffinity chromatography. The purified K-raWe describe methods for the production of lipid-modified ras proteins using the insect/baculovirus expression system. Protocols for labeling these proteins with precursors of these modifications, e.g., [ methyl - 3 H]methionine, [ 3 H]mevalonic acid, and [ 3 H]palmitate, are given. In addition, we detail the purification of the proteins using ion-exchange chromatography and immunoaffinity chromatography. The purified K-raWe describe methods for the production of lipid-modified ras proteins using the insect/baculovirus expression system. Protocols for labeling these proteins with precursors of these modifications, e.g., [ methyl - 3 H]methionine, [ 3 H]mevalonic acid, and [ 3 H]palmitate, are given. In addition, we detail the purification of the proteins using ion-exchange chromatography and immunoaffinity chromatography. The purified K-raWe describe methods for the production of lipid-modified ras proteins using the insect/baculovirus expression system. Protocols for labeling these proteins with precursors of these modifications, e.g., [ methyl - 3 H]methionine, [ 3 H]mevalonic acid, and [ 3 H]palmitate, are given. In addition, we detail the purification of the proteins using ion-exchange chromatography and immunoaffinity chromatography. The purified K-ras4B (Val-12) protein has been subjected to mass spectrometry using electrospray ionization in conjunction with an API source. The data obtained support the proposed pathway of carboxyl-terminal modification of ras proteins, i.e., farnesylation and removal of three carboxyl-terminal amino acids. However, it appears that the insect/baculovirus expression system is not able fully to carboxyl-methylate the ras proteins.B (Val-12) protein has been subjected to mass spectrometry using electrospray ionization in conjunction with an API source. The data obtained support the proposed pathway of carboxyl-terminal modification of ras proteins, i.e., farnesylation and removal of three carboxyl-terminal amino acids. However, it appears that the insect/baculovirus expression system is not able fully to carboxyl-methylate the ras proteins.B (Val-12) protein has been subjected to mass spectrometry using electrospray ionization in conjunction with an API source. The data obtained support the proposed pathway of carboxyl-terminal modification of ras proteins, i.e., farnesylation and removal of three carboxyl-terminal amino acids. However, it appears that the insect/baculovirus expression system is not able fully to carboxyl-methylate the ras proteins.B (Val-12) protein has been subjected to mass spectrometry using electrospray ionization in conjunction with an API source. The data obtained support the proposed pathway of carboxyl-terminal modification of ras proteins, i.e., farnesylation and removal of three carboxyl-terminal amino acids. However, it appears that the insect/baculovirus expression system is not able fully to carboxyl-methylate the ras proteins.