Intein-mediated cyclization of randomized peptides in the periplasm of Escherichia coli and their extracellular secretion.
Split-inteins can be used to generate backbone cyclized peptide as a source of new bioactive molecules. In this work we show that cysteine-mediated splicing can be performed in the oxidative environment of the periplasm of Escherichia coli. Cyclization of the TEM-1 beta-lactamase and of small randomized peptides was demonstrated using an artificially permuted version of the DnaB mini-intein from Synechocystis sp. PCC6803 strain fused to a signal sequence. For small peptides, a signal sequence that promotes cotranslational translocation had to be used. Efficient backbone cyclization was observed for more than 50% of combinatorial peptides featuring a fully randomized sequence inserted between a serine and glycine that are necessary for fast splicing. Furthermore, by coexpressing a mutant of the pIV outer membrane pore protein of fd bacteriophage, we showed that peptides can diffuse in the extracellular medium. These results open new routes for searching compounds acting on new targets such as exported and membrane proteins or pathogen microorganisms.
Biochimie et Genetique Moleculaire Bacterienne, Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium.
Geneviève
G
Deschuyteneer
Stéphanie
S
Garcia
Benjamin
B
Michiels
Bruno
B
Baudoux
Hervé
H
Degand
Pierre
P
Morsomme
Patrice
P
Soumillion
D001483:Base Sequence; D003500:Cyclization; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D047668:Inteins; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D019154:Protein Splicing; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism
D019151:Peptide Library; D010456:Peptides, Cyclic; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D003545:Cysteine