Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins.

Abstract

The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage X in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p2l*, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.Eukaryotic organisms, from yeast (1, 2) to man (3-7), possess a family of evolutionarily conserved genes, designated ras. These genes were originally identified as part of the oncogenic sequences of certain strains of acute transforming retroviruses (8). Recent evidence indicates that cellular ras genes acquire transforming properties by single point mutations within their coding sequences (9)(10)(11)(12)(13)(14)(15)(16)(17)(18) (18,(20)(21)(22)(23) has facilitated their molecular characterization. Yet, little is known regarding the mechanisms by which their gene products induce malignant transformation, and even less is known regarding their role in the normal cell.ras genes code for a group of highly related proteins of 189 amino acid residues designated p21. p21 proteins were originally identified by immunoprecipitation of proteins from retrovirus-infected cells with sera obtained from tumor-bearing animals (24). Scolnick and co-workers demonstrated that normal and transforming p21 proteins bind GTP and GTP derivatives such as GDP and dGTP (25,26). Under similar experimental conditions those p21 proteins containing a threonine residue in position 59 (v-rasH and v-rasK p21s) also exhibit a threonine-specific protein kinase activity (26,27).Unveiling the biochemical role that ras gene products play in normal cellular growth and differentiation, as well as in malignant transformation, requires the availability of significant amounts of purified p21 proteins. In this report, we describe the utilization of bacterial expression vectors for the synthesis of large quantities of both normal and transforming p21 proteins in Escherichia coli and the purification of these protein...

Identification and biochemical characterization of p70TRK, product of the human TRK oncogene.

“…and pEV-vrf-1 containing the bacteriophage X PL promoter were propagated in Escherichia coli DH5 cells previously transformed with the low-copy-number plasmid pRK248cIts that encodes a temperature-sensitive phage X cI repressor (18,19). The construction of the TRK expression plasmids pGM1 and pGM2 is outlined in Fig.…”

Epidermal growth factor, but not hepatocyte growth factor, suppresses the apoptosis induced by transforming growth factor‐beta in fetal hepatocytes in primary culture

“…Hybridizations were performed as previously described [7]. ¢t-Fetoprotein [18], albumin [19] and 18S ribosomal [20] lation, whereas v-fos [21], H-ras, [22] and HNF-4 [23] were labeled by random priming reaction. Serial hybridizations with the different probes were performed succesively.…”

Conformational alterations detected by circular dichroism induced in the normal ras p21 protein by activating point mutations at position 12, 59, or 61

“…This model postulates that amino acids around position 12 are located in a loop related to the interaction of the phosphate groups of the GTP molecule, between a hydrophobic P-structure (amino acids 5-10) and an amphiphilic a-helix (amino acids [16][17][18][19][20][21][22][23][24][25][26][27][28][29]. Amino acids 59 and 61 would be located in a P-turn which comprises residues 58-61, between a fl-structure (amino acids 50-57) and an or-helix (amino acids 61 -74), possibly implicated in the interaction with Mgz+ ions [18].…”