Protein Engineering Protocols - Mycobacteriology research center

228 Fujita et al.Fig. 3. (Continued)to retain the activity of an enzyme displayed on a ribosome (22,23). Thus, theamount of mRNA isolated by the ribosome display system is influenced by thelength of the spacer and the secondary structure of its 3′-end (6,18,24). We introduceda long and a short spacer sequence, separately, at the 3′-terminal end ofthe ORF and compared the effects of the two spacers on translation and selection.We found that the long spacer (of 404 amino acids, encoded by full-lengthgene III, in its entirety, from the filamentous phage M13) was not suitable fortranslation and selection in our system. Therefore, in all of our experiments, weused a fragment of 121 amino acids as the spacer.3.1.2. Construction of pStAv-R and pGST-R PlasmidsTo confirm the validity of the proposed method and the potential usefulnessof RIDS, we introduced the gene for streptavidin or GST into a DNA sequencelibrary as the model study (Fig. 3A). These proteins have frequently been fusedto newly discovered proteins and/or the molecules with which they interact forthe successful functional characterization of such newly discovered, fused proteins(25–28). Streptavidin and GST bind to biotin and glutathione, respectively,with high affinity and specificity. Thus, it should be possible to isolateand characterize the protein–ribosome–mRNA ternary complex with ease.DNA encoding for residues of streptavidin and GST were excised from theplasmids, pSTA (a gift from Prof. M. Sisido, Okayama University) and pGEX-4T-3 (Amersham Biosciences), respectively. These fragments were amplifiedby PCR and ligated separately into the XbaI/NheI sites in the pLRS plasmid.The sequences of the DNA construct encoding streptavidin or GST are shownin Fig. 3B,C.