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PMID: 6285367 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 79 ·No. 7 ·1982-04-00 ·Pages 2152-6

Gilmer TM, Parsons JT, Erikson RL

Abstract

We have constructed plasmids that direct the synthesis of the Rous sarcoma virus transforming gene (src) product (p60src) in Escherichia coli. A 203-base-pair lac promoter-operator DNA encoding the first eight amino acids of beta-galactosidase was ligated to the 5' end of the src gene from the Prague A strain of Rous sarcoma virus (PrA-RSV) which had been cloned in pBR325. Antiserum, from a tumor-bearing rabbit, directed against pp60src was used to screen bacteria containing the recombinant plasmid for a protein of approximately 60,000 daltons, and several colonies producing a protein immunologically related to pp60src were detected. Partial proteolytic cleavage analysis revealed that the src-related protein produced in bacteria is structurally similar to pp60src immunoprecipitated from PrA-RSV-infected chicken cells. Partially purified src protein from E. coli can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. Tryptic phosphopeptide analysis demonstrated that the catalytic subunit phosphorylated a serine-containing tryptic peptide in the bacterial src protein that comigrated with the phosphoserine-containing tryptic peptide of pp60src immunoprecipitated from 32P-labeled PrA-RSV-infected chicken cells.

MeSH Terms
Avian Sarcoma Viruses/genetics Cell Transformation, Viral DNA, Recombinant/metabolism Escherichia coli/genetics Immunosorbent Techniques Peptides/analysis Plasmids Viral Proteins/analysis,genetics beta-Galactosidase/genetics,metabolism
Chemicals
DNA, Recombinant Peptides Viral Proteins beta-Galactosidase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gilmer T M
Parsons J T
Erikson R L
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28 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.79.7.2152
Published
1982-04-00
Pages
2152-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC346148
Subset
IM
Grants
NIAID NIH HHS · AI 07209 · United States
NCI NIH HHS · CA-15823 · United States
NCI NIH HHS · CA-21117 · United States
Databases
GENBANK
J02556
External Links
PubMed source DOI: 10.1073/pnas.79.7.2152 PMC Full Text View journal details
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