TY - JOUR
T1 - Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12
AU - Jacobs, W. R.
AU - Docherty, M. A.
AU - Curtiss, R.
AU - Clark-Curtiss, J. E.
PY - 1986
Y1 - 1986
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0022532583&partnerID=8YFLogxK
U2 - 10.1073/pnas.83.6.1926
DO - 10.1073/pnas.83.6.1926
M3 - Article
C2 - 2869492
AN - SCOPUS:0022532583
SN - 0027-8424
VL - 83
SP - 1926
EP - 1930
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 6
ER -