Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc\'s protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc\'s intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody\'s solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a \"physiological test tube\" that not only reports secretory cargo\'s high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.

Intracellular protein crystallization occurs under various physiological and pathological settings, yet the underlying cellular processes remain enigmatic. After validating individual crystallization events using cellular proteins that readily crystallize in the ER (NEU1), cytosol (crystallin-γD mutant) or nucleus (CLC protein), I demonstrate three independent crystallization events can take place concurrently in different subcellular compartments of a single cell without compromising cell viability. By co-expressing NEU1 and previously reported two human monoclonal antibodies that undergo crystallization and liquid-liquid phase separation in the ER, I additionally demonstrate two independent phase separation events can be simultaneously induced in the ER lumen of a single cell without mixing or interfering each other\'s phase separation behaviors. Intracellular protein crystallization thus takes place in a crowded physiological cellular environment and does not require high protein purity. Furthermore, I report a simple method to increase the yield of intracellular protein crystals by treating the cells with a topoisomerase II inhibitor that blocks cell division without preventing cell size growth. This study not only presents accessible model tools for studying cryptic in vivo protein crystallization events, but also paves a way toward establishing the intracellular protein crystallization as a novel platform for recombinant protein expression and purification.