Samples were heated at 95?C for 10?min in an oil bath. polymer lengths are generated using atom transfer radical polymerization. Charged polymers increase conjugate solubility in ammonium sulfate and completely prevent precipitation even at 100% saturation. Atomistic molecular dynamic simulations show the impact is driven by an anti-polyelectrolyte effect from zwitterionic polymers. Uncharged polymers exhibit polymer length-dependent decreased solubility. The differences in salting-out are Rabbit polyclonal to TrkB then used to simply purify mixtures of conjugates and native proteins into single species. Increasing Rubusoside protein solubility in salt solutions through polymer conjugation could lead to many new applications of protein-polymer conjugates. 1.4) to 38.7?kDa (1.9) for pCBMA and from 17.5?kDa (1.7) to 85.4?kDa (1.7) for pOEGMA (Table?1 and Supplementary Figs.?3 and 4). Effect of polymer length on conjugate solubility Native Lyz, Lyz-initiator, and LyzCpolymer conjugates were subjected to precipitation by ammonium sulfate at pH 7.0 to determine their salting-out points (Fig.?2). Lyz has been shown to salt-out as predicted by the anion Hofmeister series at basic pH values and high ionic strength, but salt-out according to the reversed anion Hofmeister series at neutral to acidic pH and moderate ionic strength42,43. Additionally, Lyz solubility can be predicted from the cation Hofmeister series when pH? ?pI (Lyz pI: ~11)44. Native Lyz, as expected22, precipitated around 60% saturated ammonium sulfate (2.5?M) (Fig.?2a, b). Lyz-initiator also precipitated around 60% saturation. A charge-preserving ATRP initiator13 was used to synthesize the Lyz-conjugates so that the positive charges on amino groups were retained after initiator attachment. Therefore, the net numbers of positive and negative charges on the protein surface were preserved after initiator attachment causing Lyz-initiator to salt-out at a similar salt concentration to native Lyz. Open in a separate window Fig. 2 Ammonium sulfate precipitation of conjugates. a, b Native Lyz, Rubusoside Lyz(5+), and LyzCpolymer conjugates. Plots are solubility (log of the supernatant protein concentration) versus ammonium sulfate percent saturation. 100% saturation corresponds to 4.1?M salt concentration. a Lyz(5+)pCBMA conjugates with DP 18, DP 32, DP 56, DP 79, and DP 91. b Lyz(5+)pOEGMA conjugates with DP 25, DP 43, DP 90, DP 105, and DP 164. pCBMA increased Lyzs solubility while pOEGMA decreased Lyzs solubility depending on DP. c, d Ammonium sulfate precipitation of native Lyz(1+), Lyz(3+), and LyzCpolymer conjugates with lower grafting densities and low/high DP. c pCBMA conjugates of Lyz(1+) DP 14, Lyz(1+) DP 44, Lyz(3+) DP 20, and Lyz(3+) DP 66. The only pCBMA conjugate that precipitated was the lowest grafting density and lowest DP. d pOEGMA conjugates of Lyz(1+) DP 9, Lyz(1+) DP 93, Lyz(3+) DP 16, and Lyz(3+) DP 57. pOEGMA length affected solubility more than grafting density. Error bars represent the standard deviations from triplicate measurements (for 20?min to pellet any precipitated protein. The protein concentration in the supernatant was measured in triplicate by the absorbance at 280?nm. The supernatant used to determine protein concentration was place back into the sample and the next solid mass of ammonium sulfate was added. The process of mixing, sitting, centrifuging, and measuring protein concentration was repeated after each ammonium sulfate addition until 100% saturation (4.1?M) was reached. The addition of ammonium sulfate increased the solution volume to 1 1.42?mL at 100% saturation. Ammonium sulfate was also performed for native protein in the presence of free pCBMA or pOEGMA. In this case, native Lyz was dissolved at 2?mg/mL (1?mL starting volume) in 50?mM NaPhos buffer, pH 7. Lyophilized pCBMA or pOEGMA was Rubusoside added to match the amount (by mass), as estimated from the BCA results, of polymer present during the precipitation experiment of Lyz(5+)pCBMA DP 91 and Lyz(5+)pOEGMA DP 164. The process of ammonium sulfate precipitation was then carried out as previously.