Selective Uptake and Refolding of Globular Proteins in Coacervate Microdroplets.
Intrinsic differences in the molecular sequestration of folded and unfolded proteins within poly(diallyldimethylammonium) (PDDA)/poly(acrylate) (PAA) coacervate microdroplets are exploited to establish membrane-free microcompartments that support protein refolding, facilitate the recovery of secondary structure and enzyme activity, and enable the selective uptake and exclusion of folded and unfolded biomolecules, respectively. Native bovine serum albumin, carbonic anhydrase, and α-chymotrypsin are preferentially sequestered within positively charged coacervate microdroplets, and the unfolding of these proteins in the presence of increasing amounts of urea results in an exponential decrease in the equilibrium partition constants as well as the kinetic release of unfolded molecules from the droplets into the surrounding continuous phase. Slow refolding in the presence of positively charged microdroplets leads to the resequestration of functional proteins and the restoration of enzymatic activity; however, fast refolding results in protein aggregation at the droplet surface. In contrast, slow and fast refolding in the presence of negatively charged PDDA/PAA droplets gives rise to reduced protein aggregation and misfolding by interactions at the droplet surface to give increased levels of protein renaturation. Together, our observations provide new insights into the bottom-up design and construction of self-assembling microcompartments capable of supporting the selective uptake and refolding of globular proteins.
Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol , Bristol BS8 1TS, United Kingdom.
Nicolas
N
Martin
Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol , Bristol BS8 1TS, United Kingdom.
Mei
M
Li
Centre for Protolife Research and Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol , Bristol BS8 1TS, United Kingdom.
Stephen
S
Mann
D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D011095:Polyethylenes / Q000737:chemistry*; D058849:Protein Refolding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D000180:Acrylic Resins; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D012710:Serum Albumin, Bovine; C006903:carbopol 940