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Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.
Proc Natl Acad Sci U S A 1983; 80(14):4432-6PN

Abstract

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Authors

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Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

6308625

Citation

Weinstock, G M., et al. "Open Reading Frame Expression Vectors: a General Method for Antigen Production in Escherichia Coli Using Protein Fusions to Beta-galactosidase." Proceedings of the National Academy of Sciences of the United States of America, vol. 80, no. 14, 1983, pp. 4432-6.
Weinstock GM, ap Rhys C, Berman ML, et al. Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. Proc Natl Acad Sci USA. 1983;80(14):4432-6.
Weinstock, G. M., ap Rhys, C., Berman, M. L., Hampar, B., Jackson, D., Silhavy, T. J., ... Zweig, M. (1983). Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. Proceedings of the National Academy of Sciences of the United States of America, 80(14), pp. 4432-6.
Weinstock GM, et al. Open Reading Frame Expression Vectors: a General Method for Antigen Production in Escherichia Coli Using Protein Fusions to Beta-galactosidase. Proc Natl Acad Sci USA. 1983;80(14):4432-6. PubMed PMID: 6308625.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. AU - Weinstock,G M, AU - ap Rhys,C, AU - Berman,M L, AU - Hampar,B, AU - Jackson,D, AU - Silhavy,T J, AU - Weisemann,J, AU - Zweig,M, PY - 1983/7/1/pubmed PY - 1983/7/1/medline PY - 1983/7/1/entrez SP - 4432 EP - 6 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 80 IS - 14 N2 - We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/6308625/Open_reading_frame_expression_vectors:_a_general_method_for_antigen_production_in_Escherichia_coli_using_protein_fusions_to_beta_galactosidase_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=6308625 DB - PRIME DP - Unbound Medicine ER -