Tolerance for random recombination of domains in prokaryotic and eukaryotic translation systems: Limited interdomain misfolding in a eukaryotic translation system

Abstract

It has been proposed that eukaryotic translation systems have a greater capacity for cotranslational folding of domains than prokaryotic translation systems, which reduces interdomain misfolding in multidomain proteins and, therefore, leads to tolerance for random recombination of domains. However, there has been a controversy as to whether prokaryotic and eukaryotic translation systems differ in the capacity for cotranslational domain folding. Here, to examine whether these systems differ in the tolerance for the random domain recombination, we systematically combined six proteins, out of which four are soluble and two are insoluble when produced in an Escherichia coli and a wheat germ cell-free protein synthesis systems, to construct a fusion protein library. Forty out of 60 two-domain proteins and 114 out of 120 three-domain proteins were more soluble when produced in the wheat system than in the E. coli system. Statistical analyses of the solubilities and the activities indicated that, in the wheat system but not in the E. coli system, the two soluble domains comprised mainly of beta-sheets tend to avoid interdomain misfolding and to fold properly even at the neighbor of the misfolded domains. These results demonstrate that a eukaryotic system permits the concomitance of a wider variety of domains within a single polypeptide chain than a prokaryotic system, which is probably due to the difference in the capacity for cotranslational folding. This difference is likely to be related to the postulated difference in the tolerance for random recombination of domains.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

“…Further, several in vitro expression systems can be primed with linear non-clonal templates generated by PCR synthesis, thus lifting the requirements for cloning and sequencing. The main limitation of the cell-free systems so far was the high cost and low yields of eukaryotic system and poor performance of prokaryotic system in folding multisubunit eukaryotic proteins [21]. …”

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

“…It has been reported that a wheat germ cell-free protein synthesis system using purified wheat embryos is suitable for synthesizing a large set of multidomain proteins simultaneously (15) and for producing large multidomain proteins that are difficult to produce using E. coli (15,16). Recently, the high-throughput synthesis of the 42 components (excluding CipA) of the C. thermocellum cellulosome using the wheat germ cell-free system was reported (17).…”

Cotranslational Folding Promotes β-Helix Formation and Avoids Aggregation In Vivo

“…For example, experiments with both bacterial and firefly luciferase have demonstrated that these nascent chains adopt conformations co-translationally that are not populated during refolding from denaturant2,3, and these co-translational conformations fold to the native state much more efficiently than the conformations populated during refolding from denaturant. However, there continues to be debate as to what extent large, multi-domain proteins can fold co-translationally in prokaryotes4–7.…”