title: Application of the "codon-shuffling" method. Synthesis and selection of de novo proteins as antibacterials.
abstract: Library-based methods of non-rational and part-rational designed de novo peptides are worthy beacons in the search for bioactive peptides and proteins of medicinal importance. In this report, we have used a recently developed directed evolution method called "codon shuffling" for the synthesis and selection of bioactive proteins. The selection of such proteins was based on the creation of an inducible library of "codon-shuffled" genes that are constructed from the ligation-based assembly of judiciously designed hexamer DNA duplexes called dicodons. Upon induction with isopropyl 1-thio-beta-D-galactopyranoside, some library members were found to express dicodon-incorporated proteins. Because of this, the host cells, in our case Escherichia coli, were unable to grow any further. The bactereostatic/lytic nature of the dicodon proteins was monitored by growth curves as well as by zone clearance studies. Transmission electron microscopy of the affected cells illustrated the extent of cell damage. The proteins themselves were overexpressed as fusion partners and subsequently purified to homogeneity. One such purified protein was found to strongly bind heparin, an indication that the interaction of the de novo proteins may be with the nucleic acids of the host cell, much like many of the naturally occurring antibacterial peptides, e.g. Buforin. Therefore, our approach may help in generating a multitude of finely tuned antibacterial proteins that can potentially be regarded as lead compounds once the method is extended to pathogenic hosts, such as Mycobacteria, for example.
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Alka
authors.initials: A
authors.lastname: Rao
authors.forename: Sidharth
authors.initials: S
authors.lastname: Chopra
authors.forename: Geeta
authors.initials: G
authors.lastname: Ram
authors.forename: Ankit
authors.initials: A
authors.lastname: Gupta
authors.forename: Anand
authors.initials: A
authors.lastname: Ranganathan
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000737:chemistry; D003062:Codon; D008969:Molecular Sequence Data; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D000900:Anti-Bacterial Agents; D003062:Codon; D011506:Proteins