Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12

W. R. Jacobs, M. A. Docherty, R. Curtiss, J. E. Clark-Curtiss

Research output: Contribution to journalArticle

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Abstract

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate β-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptide produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecular was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the β-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amounts than when expressed from the S. mutans asd promoter. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential anti-leprosy drugs.

Original languageEnglish (US)
Pages (from-to)1926-1930
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number6
DOIs
StatePublished - Jan 1 1986
Externally publishedYes

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Mycobacterium leprae
Citrate (si)-Synthase
Streptococcus mutans
Escherichia coli
Peptides
Genes
DNA
Leprosy
Mutation
Aspartate-Semialdehyde Dehydrogenase
Genomic Library
Southern Blotting
Genetic Promoter Regions
Sodium Dodecyl Sulfate
Early Diagnosis
Proteins
Vaccines
Clone Cells
Antigens
Pharmaceutical Preparations

ASJC Scopus subject areas

  • General

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Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12. / Jacobs, W. R.; Docherty, M. A.; Curtiss, R.; Clark-Curtiss, J. E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 83, No. 6, 01.01.1986, p. 1926-1930.

Research output: Contribution to journalArticle

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