Study on protein network using novel ribosome-display method

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For the development of ribosome display method, the stability of ternary complex consisting of ribosome, mRNA and polypeptide is very crucial. To increase the stability, influence of insertion of spacer sequence at the C-terminus of HyHEL10scFv (lysozyme specific monoclonal antibody) was examined. It turned out that the introduction of translation arrest sequence of Sec M, F X X X X W I X X X X G I R A G P, caused efficient recovery of mRNA during the selection of ribosome-display procedure. It is likely that this was due to the stability of the ribosome complex by the SecM introduction. Moreover, it indicates that PURESYSTEM is very useful because no tmRNA which conducts ribosome recycling process, exists in the system, which completely different from the system by crude cell extract.. Omission experiment of particular factors (EF, ribosome, and mRNA) from the system towards HyHEL110 mRNA among excess amount of DHFR mRNA showed that the recovery of mRNA is actually mediated via ribosomal ternary complex. Moreover we succeeded in the concentration of a target molecule more than12000-fold in a single round selection. It is thought that this shows the effectiveness of PURESYSTEM for ribosome-display because only less than 1000 times concentration efficiency in the ribosome-disply method using cell-extract has been reported so far.