ABSTRACT
The expression vectors pINIII-A and pINIII (lpp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini. Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies. After induction with isopropyl-beta-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein. The majority of the protein was found in the postsonication low-speed centrifugation pellet. The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays.
Subject(s)
Escherichia coli/genetics , Genes , Kirsten murine sarcoma virus/genetics , Oncogene Proteins, Viral/genetics , Oncogenes , Proto-Oncogene Proteins/genetics , Sarcoma Viruses, Murine/genetics , Viral Proteins/genetics , DNA Restriction Enzymes , Genetic Vectors , Humans , Oncogene Protein p21(ras) , Plasmids , Proto-Oncogene Proteins p21(ras)Subject(s)
Biotin/metabolism , Citric Acid Cycle , Tumor Virus Infections/metabolism , Animals , Cell Line , Fibroblasts/metabolism , Mice , Molecular Weight , Simian virus 40ABSTRACT
A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G-100 at pH 2 followed by either Sephadex G-25 or BioGel P-2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat-insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive. Its biological mode of action is presently obscure. While it does not effect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15-0.30% calf serum) medium and, when added to "stationary phase" cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.
