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The mycobacterium tuberculosis reca intein can be used in an orftrap to select for open reading frames
Author(s) -
Daugelat Sabine,
Jacobs William R.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.3.644
Subject(s) - intein , biology , genetics , orfs , open reading frame , plasmid , rna splicing , protein tag , gene , fusion protein , peptide sequence , recombinant dna , rna
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.