Protein Engineering Protocols - Mycobacteriology research center

Ribosome-Inactivation Display System 225Fig. 2. (Continued)The linker fragment, encoding NheI, the glycine/serine-rich sequence ofgene III of M13, NcoI, BamHI, PstI, and XhoI in sequence from upstream, wassynthesized by a DNA synthesizer and was inserted into pET30a plasmid byXbaI/XhoI downstream of the T7 promoter. DNA coding for RTA was preparedby PCR from pUTA (a gift from Prof. J. Robertus, University of Texas) andinserted downstream of the glycine/serine-rich sequence by NcoI/BamHI. DNAcoding for the spacer was derived from gene III of M13 and inserted downstreamof RTA by BamHI/PstI. The sequence of the DNA construct, withoutinserting the protein library for RIDS, is shown in Fig. 2B.The T7 promoter and the 71-mer sequence between the promoter and thefirst ATG of the protein library were derived from the sequence of pET30a.A flexible linker upstream of RTA was indispensable to allow nascent proteins tofold into their natural three-dimensional structures to eliminate steric hindrancebetween the protein library and the downstream RTA. Previous investigationsdemonstrated that the yield of a selected protein/mRNA is strongly dependenton the length, composition, and sequence of such a linker (6,16,17). In thisstudy, we used a glycine/serine-rich fragment of 44 amino acids as the linker.It is important to eliminate the steric hindrance of RTA, because, in the RIDS,stabilization of the mRNA–ribosome–protein complex was achieved by introducingthe gene for RTA. In addition to the linker, a spacer at the 3′-terminal endof the open-reading frame (ORF) was also important. The spacer, which wasrequired as an anchor to tether the ribosome, must be of appropriate length sothat it can occupy the long tunnel of the ribosome (18,19) and allow the nascentRTA to fold correctly without any steric hindrance (20,21). It has been reportedthat a spacer of at least 20 to 30 amino acids at the carboxyl terminus is required