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. 1999 Mar;8(3):644–653. doi: 10.1110/ps.8.3.644

The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames.

S Daugelat 1, W R Jacobs Jr 1
PMCID: PMC2144272  PMID: 10091667

Abstract

The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.

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Selected References

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  1. Adler H. I., Fisher W. D., Cohen A., Hardigree A. A. MINIATURE escherichia coli CELLS DEFICIENT IN DNA. Proc Natl Acad Sci U S A. 1967 Feb;57(2):321–326. doi: 10.1073/pnas.57.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chong S., Mersha F. B., Comb D. G., Scott M. E., Landry D., Vence L. M., Perler F. B., Benner J., Kucera R. B., Hirvonen C. A. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene. 1997 Jun 19;192(2):271–281. doi: 10.1016/s0378-1119(97)00105-4. [DOI] [PubMed] [Google Scholar]
  3. Chong S., Shao Y., Paulus H., Benner J., Perler F. B., Xu M. Q. Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem. 1996 Sep 6;271(36):22159–22168. doi: 10.1074/jbc.271.36.22159. [DOI] [PubMed] [Google Scholar]
  4. Chong S., Xu M. Q. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J Biol Chem. 1997 Jun 20;272(25):15587–15590. doi: 10.1074/jbc.272.25.15587. [DOI] [PubMed] [Google Scholar]
  5. Cooper A. A., Chen Y. J., Lindorfer M. A., Stevens T. H. Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision. EMBO J. 1993 Jun;12(6):2575–2583. doi: 10.1002/j.1460-2075.1993.tb05913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis E. O., Jenner P. J., Brooks P. C., Colston M. J., Sedgwick S. G. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell. 1992 Oct 16;71(2):201–210. doi: 10.1016/0092-8674(92)90349-h. [DOI] [PubMed] [Google Scholar]
  7. Davis E. O., Jenner P. J. Protein splicing--the lengths some proteins will go to. Antonie Van Leeuwenhoek. 1995;67(2):131–137. doi: 10.1007/BF00871208. [DOI] [PubMed] [Google Scholar]
  8. Davis E. O., Sedgwick S. G., Colston M. J. Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. J Bacteriol. 1991 Sep;173(18):5653–5662. doi: 10.1128/jb.173.18.5653-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derbyshire V., Wood D. W., Wu W., Dansereau J. T., Dalgaard J. Z., Belfort M. Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11466–11471. doi: 10.1073/pnas.94.21.11466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duan X., Gimble F. S., Quiocho F. A. Crystal structure of PI-SceI, a homing endonuclease with protein splicing activity. Cell. 1997 May 16;89(4):555–564. doi: 10.1016/s0092-8674(00)80237-8. [DOI] [PubMed] [Google Scholar]
  11. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  12. Gu H. H., Xu J., Gallagher M., Dean G. E. Peptide splicing in the vacuolar ATPase subunit A from Candida tropicalis. J Biol Chem. 1993 Apr 5;268(10):7372–7381. [PubMed] [Google Scholar]
  13. Hatfull G. F., Sarkis G. J. DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol Microbiol. 1993 Feb;7(3):395–405. doi: 10.1111/j.1365-2958.1993.tb01131.x. [DOI] [PubMed] [Google Scholar]
  14. Hirata R., Anraku Y. Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing. Biochem Biophys Res Commun. 1992 Oct 15;188(1):40–47. doi: 10.1016/0006-291x(92)92347-z. [DOI] [PubMed] [Google Scholar]
  15. Hirata R., Ohsumk Y., Nakano A., Kawasaki H., Suzuki K., Anraku Y. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Apr 25;265(12):6726–6733. [PubMed] [Google Scholar]
  16. Hodges R. A., Perler F. B., Noren C. J., Jack W. E. Protein splicing removes intervening sequences in an archaea DNA polymerase. Nucleic Acids Res. 1992 Dec 11;20(23):6153–6157. doi: 10.1093/nar/20.23.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kane P. M., Yamashiro C. T., Wolczyk D. F., Neff N., Goebl M., Stevens T. H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 1990 Nov 2;250(4981):651–657. doi: 10.1126/science.2146742. [DOI] [PubMed] [Google Scholar]
  18. Kawasaki M., Nogami S., Satow Y., Ohya Y., Anraku Y. Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence. J Biol Chem. 1997 Jun 20;272(25):15668–15674. doi: 10.1074/jbc.272.25.15668. [DOI] [PubMed] [Google Scholar]
  19. Mengaud J., Vicente M. F., Chenevert J., Pereira J. M., Geoffroy C., Gicquel-Sanzey B., Baquero F., Perez-Diaz J. C., Cossart P. Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect Immun. 1988 Apr;56(4):766–772. doi: 10.1128/iai.56.4.766-772.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mills K. V., Lew B. M., Jiang S., Paulus H. Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3543–3548. doi: 10.1073/pnas.95.7.3543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Perler F. B., Comb D. G., Jack W. E., Moran L. S., Qiang B., Kucera R. B., Benner J., Slatko B. E., Nwankwo D. O., Hempstead S. K. Intervening sequences in an Archaea DNA polymerase gene. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5577–5581. doi: 10.1073/pnas.89.12.5577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perler F. B., Davis E. O., Dean G. E., Gimble F. S., Jack W. E., Neff N., Noren C. J., Thorner J., Belfort M. Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res. 1994 Apr 11;22(7):1125–1127. doi: 10.1093/nar/22.7.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Perler F. B., Olsen G. J., Adam E. Compilation and analysis of intein sequences. Nucleic Acids Res. 1997 Mar 15;25(6):1087–1093. doi: 10.1093/nar/25.6.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pietrokovski S. Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Sci. 1994 Dec;3(12):2340–2350. doi: 10.1002/pro.5560031218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pietrokovski S. Modular organization of inteins and C-terminal autocatalytic domains. Protein Sci. 1998 Jan;7(1):64–71. doi: 10.1002/pro.5560070106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shao Y., Kent S. B. Protein splicing: occurrence, mechanisms and related phenomena. Chem Biol. 1997 Mar;4(3):187–194. doi: 10.1016/s1074-5521(97)90287-8. [DOI] [PubMed] [Google Scholar]
  27. Shub D. A., Goodrich-Blair H. Protein introns: a new home for endonucleases. Cell. 1992 Oct 16;71(2):183–186. doi: 10.1016/0092-8674(92)90345-d. [DOI] [PubMed] [Google Scholar]
  28. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H., Hatfull G. F. New use of BCG for recombinant vaccines. Nature. 1991 Jun 6;351(6326):456–460. doi: 10.1038/351456a0. [DOI] [PubMed] [Google Scholar]
  30. Telenti A., Southworth M., Alcaide F., Daugelat S., Jacobs W. R., Jr, Perler F. B. The Mycobacterium xenopi GyrA protein splicing element: characterization of a minimal intein. J Bacteriol. 1997 Oct;179(20):6378–6382. doi: 10.1128/jb.179.20.6378-6382.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu M. Q., Perler F. B. The mechanism of protein splicing and its modulation by mutation. EMBO J. 1996 Oct 1;15(19):5146–5153. [PMC free article] [PubMed] [Google Scholar]
  32. Xu M. Q., Southworth M. W., Mersha F. B., Hornstra L. J., Perler F. B. In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell. 1993 Dec 31;75(7):1371–1377. doi: 10.1016/0092-8674(93)90623-x. [DOI] [PubMed] [Google Scholar]

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