A type of filamentous protein assembly known as agglomerates appear to offer advantages in intracellular engineering and synthetic biology and little to no deleterious effects on cellular fitness, according to a recent paper.  This paves the way to using agglomerates in biomanufacturing to, for example, track protein expression, create molecular sensors for bioprocessing, or design them as in-cell scaffolds in metabolic bioengineering

These novel assemblies haven’t yet been broadly recognized, however, because they are difficult to distinguish from protein aggregates. Yet, that may now change, with the development of a novel assay by researchers led by Emmanuel Levy, PhD, a professor at the University of Geneva, and formerly associate professor at the Weizmann Institute of Science, together with colleagues from those universities and the Molecular Biology Institute of Barcelona, ETH Zurich, and University of Zurich.

Their in-cell agglomerate dissolution assay distinguishes agglomerates from misfolded aggregates in a yeast proteome based on whether the expression of a wild-type protein prevents puncta formation. Puncta, they found, are associated with aggregates but not with agglomerates. This method, therefore, makes it possible to classify protein assemblies in vivo, on a continuum between agglomeration and aggregation.

Agglomerates vs aggregates

Agglomerates result from mutations that cause the assembly of folded protein subunits to interact with themselves. Their benefit, compared to aggregates (which are formed by misfolded protein subunits), is that they “retain their folded state, do not co-localize with the proteostasis machinery, and are not ubiquitinated. They cause no detectable growth defects,” the scientists reported.

For bioprocessing, “These properties mean they are compatible with applications requiring bio-orthogonality, in which a new engineered function is desired while intracellular side effects are minimized,” first author Tal Levin, PhD, postdoctoral fellow, Weizmann Institute of Science, tells GEN.

“Agglomerates are dissolved when wild-type subunits are co-expressed and incorporated into the assemblies,” the scientists pointed out. This capability suggests their presence can be modulated in bioengineering and, possibly, therapeutic applications,” Levin elaborates. “For example, in a follow-up study, the induction of agglomerates by small molecules served as a novel PROTAC-like pharmacological modality.

Key advantage: Reversibility

“A practical benefit,” Levin continues, “is that agglomerations can be reversible. In nature, many enzymes form agglomerates in a condition-dependent manner. This enables agglomeration to act as a regulatory mechanism.”

For example, Levin says, a team headed by Patrick Stoddard, PhD, now associate director, New Culture, while at Harvard University, showed “the agglomerate form of Glk1 is inactive, while the soluble form is active. Under high glucose conditions, the excess enzyme agglomerates slow enzymatic activity and prevent accumulation of toxic glycolysis intermediaries.”

Practically speaking, Levin says, engineers can design an enzyme “to agglomerate in low pH and become temporarily inactive…rather than creating a bad byproduct.” This functionality, therefore, is conducive to protein bioprocessing.

In biomanufacturing, therefore, agglomerates can be used to increase local protein concentrations because, “unlike aggregates, an enzyme can still be active while in its agglomerate form.”