Study of cell‐free protein synthesis in aqueous two‐phase systems

Abstract

Protein synthesis in an aqueous two-phase system is reported here as a novel type of extractive bioconversion. Translation of BMV RNA was studied using either rabbit reticulocyte lysate or wheat-germ extract in aqueous dextran-PEG systems. Both polymers inhibited protein synthesis and the translation system partitioned into both phases. In the rabbit reticulocyte two-phase system, protein synthesis reached 30% of that in free solution, while in wheat-germ extract it was 60% of that in free solution. Protein was found mainly in the dextran (lower) phase but its partitioning was related to the polymer concentration, and molecular weight, as well as the ionic strength of the system. Protein synthesis was highly influenced by PEG concentration, potassium chloride concentration, and dextran molecular weight.

Multiphase Water-in-Oil Emulsion Droplets for Cell-Free Transcription–Translation

“…9,10 Others demonstrated that transcription–translation (T/T) can be carried out in vesicles 11 and in bulk aqueous two-phase systems (ATPS). 12,13 By combining these approaches, it should be possible to produce genetically encoded proteins that distribute between coexisting phases as a model for cellular microcompartments.…”

“…One approach toward gaining insight into the organizational features of cellular life is to construct mimetic systems in the laboratory that display similar organization and behavior. , Several recent studies have defined a set of chemical conditions that give rise to two coexisting aqueous phases in lipid vesicles. , Others demonstrated that transcription–translation (T/T) can be carried out in vesicles and in bulk aqueous two-phase systems (ATPS). , By combining these approaches, it should be possible to produce genetically encoded proteins that distribute between coexisting phases as a model for cellular microcompartments.…”

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering

“…Moving on from transcription, translation has also been studied within phase-separated compartments. Cell-free mRNA translation processes in PEG/dextran ATPS can occur unimpeded, and are of interest to large scale production of proteins in bioindustrial applications (Marszal et al 1995). Targeted mRNA suppression and translation has also been observed to occur within a membraneless organelle inside eukaryotic cells (Reinkemeier et al 2019).…”

Toward long-lasting artificial cells that better mimic natural living cells

“…Compartments can even be made without a membrane at all [20]. Neither aqueous two-phase systems [21,22], polyelectrolyte polymer containing coacervates [23], hydrogels [24], microfluidic chip-based compartments [25,26], nor bead-based systems [27] contain a membrane.…”