Construction of Plasmids for Expression of Rous Sarcoma Virus Transforming Protein, P60src, in Escherichia Coli

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1982 Apr 1

PMID 6285367

Citations 17

Authors

T M Gilmer

J T Parsons

R L Erikson

Affiliations

Soon will be listed here.

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.

Citing Articles

P85gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity.

Kloetzer W, Maxwell S, Arlinghaus R Proc Natl Acad Sci U S A. 1983; 80(2):412-6.

PMID: 6601272 PMC: 393387. DOI: 10.1073/pnas.80.2.412.

Development of anti-pp60src serum with antigen produced in Escherichia coli.

Gilmer T, Erikson R J Virol. 1983; 45(1):462-5.

PMID: 6401819 PMC: 256431. DOI: 10.1128/JVI.45.1.462-465.1983.

Expression of the chicken c-src gene in COS cells.

Gilmer T Mol Cell Biol. 1984; 4(5):846-51.

PMID: 6328271 PMC: 368826. DOI: 10.1128/mcb.4.5.846-851.1984.

Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli.

Stein R, Robinson P, Scolnick E J Virol. 1984; 50(2):343-51.

PMID: 6323735 PMC: 255626. DOI: 10.1128/JVI.50.2.343-351.1984.

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

Purchio A, Wells S, Collett M Mol Cell Biol. 1983; 3(9):1589-97.

PMID: 6314123 PMC: 370012. DOI: 10.1128/mcb.3.9.1589-1597.1983.

References

Clewell D, Helinski D . Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969; 62(4):1159-66. PMC: 223628. DOI: 10.1073/pnas.62.4.1159. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Davies J, Jacob F . Genetic mapping of the regulator and operator genes of the lac operon. J Mol Biol. 1968; 36(3):413-7. DOI: 10.1016/0022-2836(68)90165-4. View

Roberts J, Roberts C . Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc Natl Acad Sci U S A. 1975; 72(1):147-51. PMC: 432259. DOI: 10.1073/pnas.72.1.147. View

Betz J, SADLER J . Tight-binding repressors of the lactose operon. J Mol Biol. 1976; 105(2):293-319. DOI: 10.1016/0022-2836(76)90113-3. View

Backman K, Ptashne M, Gilbert W . Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976; 73(11):4174-8. PMC: 431373. DOI: 10.1073/pnas.73.11.4174. View

Cleveland D, Fischer S, Kirschner M, Laemmli U . Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977; 252(3):1102-6. View

Bechtel P, Beavo J, KREBS E . Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977; 252(8):2691-7. View

Brugge J, Erikson R . Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977; 269(5626):346-8. DOI: 10.1038/269346a0. View

10.

Bolivar F, Rodriguez R, Betlach M, Boyer H . Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977; 2(2):75-93. DOI: 10.1016/0378-1119(77)90074-9. View

11.

Collett M, Erikson R . Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978; 75(4):2021-4. PMC: 392475. DOI: 10.1073/pnas.75.4.2021. View

12.

Fowler A, Zabin I . Amino acid sequence of beta-galactosidase. VIII. Sequence of the NH2-terminal segment, CNBr peptides 1 to 9, residues 1 to 377. J Biol Chem. 1978; 253(15):5505-9. View

13.

Brugge J, Erikson E, Collett M, Erikson R . Peptide analysis of the transformation-specific antigen from avian sarcoma virus-transformed cells. J Virol. 1978; 26(3):773-82. PMC: 525902. DOI: 10.1128/JVI.26.3.773-782.1978. View

14.

Levinson A, Oppermann H, LEVINTOW L, Varmus H, Bishop J . Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell. 1978; 15(2):561-72. DOI: 10.1016/0092-8674(78)90024-7. View

15.

Rubsamen H, Friis R, Bauer H . Src Gene product from different strains of avian sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus. Proc Natl Acad Sci U S A. 1979; 76(2):967-71. PMC: 383109. DOI: 10.1073/pnas.76.2.967. View

16.

Collett M, Erikson E, Erikson R . Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979; 29(2):770-81. PMC: 353209. DOI: 10.1128/JVI.29.2.770-781.1979. View

17.

Erikson R, Collett M, Erikson E, Purchio A . Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc Natl Acad Sci U S A. 1979; 76(12):6260-4. PMC: 411843. DOI: 10.1073/pnas.76.12.6260. View

18.

Bolivar F, Backman K . Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979; 68:245-67. DOI: 10.1016/0076-6879(79)68018-7. View

19.

Roberts T, Lauer G . Maximizing gene expression on a plasmid using recombination in vitro. Methods Enzymol. 1979; 68:473-82. DOI: 10.1016/0076-6879(79)68036-9. View

20.

Maxam A, Gilbert W . Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980; 65(1):499-560. DOI: 10.1016/s0076-6879(80)65059-9. View