High-Level Expression in Escherichia coli of Enzymatically Active Harvey Murine Sarcoma Virus p21 ras Protein

The v-rasH oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton p21 protein which has been expressed at a high level as a fusion protein in Escherichia coli. We have purified the p21 to over 90% in purity without the use of any detergent or protein denaturant. The purified p21 possesses full biochemical activities of GTP/GDP binding, autokinase, and GTPase. Scatchard analysis indicates a single class of binding sites with Kd values of 0.83 x 10-8 M for GTP and 1.0 x 10-8 M for GDP. The binding site can be specifically labeled with a [3H]GTP photoaffinity analog, P3-(4-azidoanilido)-5' GTP. To probe for the active center of p21, we used a battery of six monoclonal antibodies to p21 to examine their effects on p21 activities. We found that only one monoclonal antibody, Y13-259, was capable of inhibiting both GTP/GDP binding and autokinase enzymatic activities, suggesting that these p21 activities are related activities conferred by a single active center within the p21 molecule. These observations together with the recent finding that microinjection of the same monoclonal antibody into NIH 3T3 cells specifically blocks p21 in vivo function (Mulcahy et al., Nature [London] 313:241, 1985) strongly suggest that p21 in vitro activities are responsible for its cellular function.

“…E. coli carrying plasmid pJLcIIrasI was grown in a fermentor (14). All p21 purification procedures were performed at 0 to 4°C.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Biochemical properties of a highly purified v-rasH p21 protein overproduced in Escherichia coli and inhibition of its activities by a monoclonal antibody.

Hattori¹,

Ulsh²,

Halliday³

et al. 1985

“…DNA transfection studies utilizing the molecularly cloned proviral DNA of Ha-MuSV and studies on the thermolability of the p21 protein of a temperature-sensitive mutant of Ki-MuSV have concluded that the p21 proteins are the gene products mediating the virusinduced malignant transformation of normal cells (4,6,20,25). p21 proteins of Ha-MuSV and Ki-MuSV possess a guanine nucleotide-binding activity and an autophosphorylation activity at the 59th threonine residue (8,9,(14)(15)(16). In contrast to the viral p21 protein, the cellular homologs are not phosphorylated due to substitution for the threonine residue with an alanine residue at the major phosphorylation site.…”

mentioning

confidence: 99%

Metabolic turnover of human c-rasH p21 protein of EJ bladder carcinoma and its normal cellular and viral homologs.

Ulsh¹,

Shih²

1984

The EJ bladder carcinoma oncogene is activated by a point mutation in the c-rasH proto-oncogene at the 12th amino acid codon. In an attempt to understand the mechanism of oncogenic activation, a comparative study was undertaken to examine the metabolic turnover and subcellular localization of the p21 protein encoded by the EJ oncogene, the viral oncogene, and its normal cellular homolog. Pulse-labeling experiments indicated that both c-ras p21 proteins were synthesized by a very similar pathway, as was observed for the viral p21 protein of Harvey murine sarcoma virus. The pro-p21 proteins were detected in free cytosol, and the processed products were associated with plasma membrane. The intracellular half-life of p21 proteins was determined by pulselabeling and chasing in the presence of excess unlabeled methionine. Although both p21 proteins of EJ and the normal c-ras genes which are not phosphorylated have a half-life of 20 h, the viral p21 protein of Harvey murine sarcoma virus which includes a phosphorylated form is much more stable in cells, having a half-life of 42 h, apparently due to phosphorylation.

“…Mutant p2l clones were inserted into the bacterial expression vector pJL6 (17), and the proteins were purified as described previously (5). As controls, mutant proteins altering amino acid 12 or amino acid 59 or both were included.…”

Section: Resultsmentioning

confidence: 99%

“…For the preparation of bacterially expressed mutant p2ls, the 0.88-kilobase Hindlll fragment from each mutant pSV2neo/ras clone was purified and ligated into the Hindlll site of the plasmid vector pJL6 (17 (5).…”

Section: Methodsmentioning

confidence: 99%

Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Clanton¹,

Lü²,

Blair³

et al. 1987

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mnutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.