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J. Biol. Chem., Vol. 280, Issue 25, 23605-23614, June 24, 2005

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Application of the "Codon-shuffling" Method

SYNTHESIS AND SELECTION OF DE NOVO PROTEINS AS ANTIBACTERIALS^*

Alka Rao, Sidharth Chopra¶, Geeta Ram, Ankit Gupta, and Anand Ranganathan||

From the Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

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--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.

Received for publication, March 21, 2005 , and in revised form, April 15, 2005.

^* This work was supported by internal grants of the International Centre for Genetic Engineering and Biotechnology and funding from the Council for Scientific and Industrial Research (to A. Rao and G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental data and Figs. S1–S8.

Both authors contributed equally to this work.

^¶ Present address: School of Medicine, Stanford University, Palo Alto, CA 94305.

^|| To whom correspondence should be addressed. Tel.: 91-11-26195007; Fax: 91-11-26162316; E-mail: anand{at}icgeb.res.in.

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				A. Rao, G. Ram, A. K. Saini, R. Vohra, K. Kumar, Y. Singh, and A. Ranganathan Synthesis and Selection of De Novo Proteins That Bind and Impede Cellular Functions of an Essential Mycobacterial Protein Appl. Envir. Microbiol., February 15, 2007; 73(4): 1320 - 1331. [Abstract] [Full Text] [PDF]