Chemical genetics reverse

A Reverse Chemical Genetic Assay Probing elF4E elF4G and... 

Hisashi Koga (ed.), Reverse Chemical Genetics, Methods in Molecular Biology, vol. 577... 

Koga, H. (2006) Establishment of the Platform for Reverse Chemical Genetics Targeting Novel Protein-Protein Interactions. Mol BioSyst, 2, 159-164. 

In reverse chemical genetics, it is crucial to synthesize proteins of interest using appropriate foreign gene expression systems and cDNA resources. Since cell-free protein synthesis systems have the potential to synthesize any desired proteins, including both native proteins and those that are toxic to cells (1), with high throughput, they can be powerful tools for this objective. We developed a cell-free protein synthesis system from Spodop-tera fm iperda 21 (S 21) insect cells, which are widely used as the host for baculovirus expression systems, and commercialized it as the Transdirect insect cell. 

We have demonstrated that this insect cell-free protein synthesis system is one of the most effective protein synthesis systems among those based on animal extracts (2). Furthermore, it has the potential to perform eukaryote-specific protein modifications such as protein W-myristoylation and prenylation (3, 4). Thus, we expect that the insect cell-free protein synthesis system will be a useful method for target protein production in the reverse chemical genetics era, as well as for postgenomic studies. In this chapter, we describe standard protocols to synthesize proteins of interest using the insect cell-free protein synthesis system. 

The progress of reverse chemical genetics research is influenced by the efficiency of generation of active recombinant proteins. In recent years, numerous systems for the expression of the recombinant proteins have been developed. Of these, the baculovirus system is considered to be the most efficient. Typically in the baculovirus system, an insect cell line (for example, Sf9) is used as a host for the expression of recombinant proteins. In the present report, we describe the novel application of Kaiko as a host in the baculovirus system for the expression of recombinant... 

To date, complex proteins with biological activity (6), for use in X-ray crystal structure analysis (7) and ELISA systems (8), and for the development of animal drugs (9) have successfully been produced by using this system. Therefore, the Kaiko-baculovirus protein production system has broad applicability across the field of reverse chemical genetics for the analysis of protein function on the basis of interactions with chemical compounds. 

Most recently, we reported small molecule arrays on photoaffinity crosslinker coated gold surfaces (17). The small molecule arrays were fabricated by photoreaction, and then analyzed by SPR imaging technique. The small molecules don t have to be modified chemically for immobilization. The small molecules, which can interact with a target protein, can be screened by this methodology. Therefore, the integration of photoaffinity small molecule array and SPR imaging technique can be the first step of reverse chemical genetics. 

---