Liquid-liquid phase separation (LLPS) is a physico-chemical phenomenon in which a solution spontaneously separates into two liquid phases, with one having a higher and one having a lower concentration of solute. Our research focused on how temperature, salt concentration, protein concentration, addition of crowding agents (eg. polyethylene glycol) and addition of a variable number of FLAG-tag repeats affects LLPS apparition of model protein nanobody 55 (Nb55). For this purpose, we used in vivo assembly cloning to create DNA constructs encoding nanobodies with 0x (without FLAG-tag), 1x, 2x and 3x repeats of the FLAG-tag. We studied the phenomenon using UV-Vis spectroscopy while cooling our samples at a rate of 0,1 °C/min. We discovered that LLPS manifests at temperatures ranging between 15 and 5 °C, if the protein has an adequate number of FLAG repeats. We confirmed a link between protein concentration and LLPS apparition, as samples with higher protein concentrations underwent LLPS at higher temperatures (around 15 °C), than those with lower protein concentrations (around 5 °C). We also established a link between the number of FLAG-tag repeats and the protein's propensity to undergo LLPS, as the proteins with 2x and 3x FLAG-tag repeats are the only ones which underwent LLPS in our studied concentration range. The addition of salt or polyethylene glycol inhibitis LLPS apparition.
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