Using the Flexible In-vitro Translation system to introduce non-proteinogenic amino acids into membrane proteins

Purpose and goal

The original goal was to adapt an in vitro synthesis system to study membrane protein biogenesis. We encountered one problem that stalled us: insufficient efficiency of ribosome targeting to the inner membrane vesicles. However, during this work we made the unanticipated discovery that the in vitro translation system can be used to follow the co-translational folding of cytoplasmic proteins. The collaboration with Prof Suga will come very handy, since we can easily introduce non-natural amino acids into the protein and thereby probe co-translational folding in great detail.

Results and expected effects

As explained above, the main result of the project is the discovery of a new way to analyze cotranslational folding of cytoplasmic proteins. We expect this to yield important new insights into protein folding.

Approach and implementation

Our approach was to optimize an in vitro protein synthesis system to work together with inverted inner membrane vesicles prepared from E. coli. We encountered one problem that we have so far been unable to solve: insufficient efficiency of ribosome targeting to the inner membrane vesicles. However, we discovered that we could tweak the in vitro synthesis system such that we can now analyze the cotranslational folding of cytoplasmic proteins. Thus, the project has serendipitously opened up a new field of research that we are now exploring.