Nucleic Acids Research Advance Access published online on February 7, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm017
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Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs
1Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan and 2CREST, Japan Science and Technology Agency (JST), c/o Cancer Institute
*To whom correspondence should be addressed. Tel: +81 3 3570 0489; Fax: +81 3 3570 0461; Email: kshiba{at}jfcr.or.jp
Received October 18, 2006. Revised December 20, 2006. Accepted January 2, 2007.
The presence of peptide motifs within the proteins provides the synthetic biologist with the opportunity to fabricate novel proteins through the programming of these motifs. Here we describe a method that enables one to combine multiple peptide motifs to generate a combinatorial protein library. With this method, a set of sense and antisense oligonucleotide primers were prepared. These primers were mixed and polymerized, so that the resultant DNA consisted of combinatorial polymers of multiple microgenes created from the stochastic assembly of the sense and antisense primers. With this motif-mixing method, we prepared a protein library from the BH1-4 motifs shared among Bcl-2 family proteins. Among the 41 clones created, 70% of clones had a stable, presumably folded expression product in human cells, which was detectable by immunohistochemistry and western blot. The proteins obtained varied with respect to both the number and the order of the four motifs. The method enables homology-independent polymerization of DNA blocks that coded motif sequences, and the frequency of each motif within a library can be adjusted in a tailor-made manner. This motif programming has a potential for creating a library with a large proportion of folded/functional proteins.
Present address: Hirohide Saito, Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
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