The avian sarcoma and leukosis viruses contain an RNA binding nucleocapsid protein, pp12, in the phosphorylated form. An Escherichia coli expression vector carrying the Rous sarcoma virus Prague C strain gene coding for this protein has been constructed using a site-directed deletion mutagenesis method to place start and stop codons into translational reading frame with the N- and C-terminal coding sequences of the protein, respectively. The protein is produced efficiently in bacteria (rp12), is soluble and readily purified. It is also indistinguishable from the unphosphorylated viral pp12 protein in its (1) migration on SDS-polyacrylamide gels, (2) reactivity with rabbit antisera directed against purified viral pp12, (3) low RNA-binding affinity, and (4) ability to serve as a substrate for in vitro phosphorylation at serine-40 by protease-activated kinase I. The ability to analyze the biochemical activities of the normal, modified, and mutant forms of this protein is an essential step in elucidating its role in the retroviral life cycle. This expression clone will be especially useful in testing for the effects of mutations before they are reconstructed into retroviral genomes for analyses.
- Oligodeoxynucleotide-directed deletion mutagenesis
- phage λ p promoter
- recombinant DNA
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