The Key to In Vitro Protein Evolution Cell-Free Translation

The Key to In Vitro Protein Evolution Cell-free Translation 

Many applications of cell-free translation rely on the correct folding of the in vitro expressed polypeptide into its three-dimensional structure, and this is a prerequisite for all protein selection systems that are based on in vitro translation. Because proteins are selected and evolved for functionality, sufficient expression and correct folding in the respective cell-free translation system are a necessity for efficient selection. An attractive advantage of using in vitro translations is that, at least in principle, any component of the reaction can be deliberately added or removed. To achieve any improvement in yield, however, separate consideration of both the actual translation and the folding is necessary. Even in optimized systems, however, translation yields are not similar for all globular proteins. 

Section III, B, 2 for discussion of E. coli RNases). Because the recognition sequences of these enzymes are different in each organism and are in many cases unknown, this problem might be solved by removing these enzymes from the translation system with immunoprecipitation. 

Moreover, it was recently shown that the deliberate removal of phosphatases from the in vitro translation mixture by immunoprecipitation results in increased protein yields (Shen et al., 1998). As proposed earlier (Jermutus et al., 1998), the elimination of one of the many causes of fast ATP and GTP depletion extends the time of synthesis, and, as a consequence, the total amount of produced protein. It should be possible to similarly remove proteases and nucleases (see above). Together with new ATP regeneration systems to keep the biochemical energy level at a high steady state (Kim and Swartz, 1999), the optimization measures mentioned should increase the level of synthesis of most proteins, and this should directly improve in vitro selection. 

Cotranslational misfolding or aggregation reduces the active fraction of synthesized proteins and might also decrease the expression yield by 

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