Protein Engineering Protocols - Mycobacteriology research center

Ribosome-Inactivation Display System 223Fig. 1. Schematic representation of the RIDS for screening functional proteins in vitro.Step 1, transcription: the gene for RTA, inserted downstream of a random protein library(or cDNA library) is transcribed by T7 RNA polymerase to yield mRNA. Step 2, translation:mRNA is translated in a rabbit reticulocyte lysate system. Step 3, inactivation: duringtranslation, because the rRNA is inactivated by folded RTA in a cis reaction, theribosome is stalled and a ribosome–mRNA–protein complex is formed. Step 4, selection:the complex of interest is bound to the corresponding affinity matrix. Unwanted complexesare removed by washing. Step 5, elution: the specific complex is dissociated fromthe matrix by elution with a buffer that contains ethylenediaminetetraacetic acid, and freemRNA is isolated. Step 6, amplification: eluted RNA is amplified by RT-PCR and theresultant cDNA is used for the next cycle or for analysis by cloning and sequencing.To evaluate the potential usefulness of RIDS, we chose streptavidin andglutathione-S-transferase (GST) as target proteins and isolated the respectiveproteins and their mRNAs using appropriate matrices that contained biotinand glutathione, respectively, as the ligands. We found that it was possible,using RIDS, to screen the functional proteins (streptavidin and GST) withoutreducing the diversity of the sequence library and without any chemical synthesis.If a complementary DNA (cDNA) library or random DNA library isintroduced into the RIDS instead of the model protein (streptavidin or GST),we will be able to select the functional protein.2. Materials1. pET30a (Novagen, Darmstadt, Germany).2. cDNA library or random DNA library.