Development of single-molecule PCR combined with cell-free protein synthesis to construct large in vitro protein library

摘要

As experimental technique for molecular evolution of protein, there are some in vivo systems such as phage display and yeast cell-surface display et al, all of which utilize growing cells. Especially the phage display method has been well developed to be commercially available as a kit. On the other hand, in vitro techniques such as ribosome display, STABLE, and in vitro virus, have been proposed although they are not widely applied. We have developing the single-molecule PCR technique followed by cell-free protein synthesis to construct very large in vitro protein library (Fig. 1). This method has following several advantageous potentials because the system is completely free from the life-maintaining constraint as compared with in vivo technique and other in vitro methods: 1) The technique is based on very simple principle, 2) The size of the library can be extended very largely, 3) The micro-array and automated manipulation of the system is easily realized, 4) The results can be obtained in short time, 5) The screening is possible with respect to not only affinity binding, but also catalytic activity, 6) Screenings of proteins toxic to living cells and of proteins involving unnatural ammo acid residue(s) are possible.