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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...

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.

The functional assay of a eukaryotic mRNA, into which additional AUG codons have been introduced by in vitro mutagenesis, shows that a translational initiation site need not necessarily be the nearest AUG codon to the 5' end of a mRNA. Hence, the sequence surrounding the AUG, and not simply its position relative to the 5' end of mRNA, appears to be important in determining initiation efficiency.

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