Example 27
The culture supernatant was firstly precipitated with ammonium sulfate (80% saturation), then dialyzed with 20 mM PBS at pH7.0. The solution was filtered with 0.45 um filter and then loaded into SP Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0. A gradient of NaCl concentration was applied as elution buffer from zero to 1M, and then elution fractions and flow-through fraction were collected to detect lysozyme activity. The fractions with lysozyme activity were pooled and analyzed by SDS-PAGE, and then concentrated for further evaluation.
Since the purified sample has two bands, the collected sample was added ammonium sulfate with a final conductivity with 180 mS/cm, and then loaded into a Phenyl Sepharose 6 Fast Flow column (GE Healthcare) equilibrated with 20 mM PBS at pH7.0 with 1.8M (NH4)2504. A concentration gradient of (NH4)2504 was applied as elution buffer from 1.8 M to zero. The fractions with lysozyme activity were collected and carried out for SDS-PAGE. The sample also contains two bands, but MS data showed both bands are target proteins. The protein concentration was determined by Qubit® Protein Assay Kit (Invitrogen, cat Q33212).
US10945449B2. Animal feed compositions and uses thereof (2021-03-16). Novozymes A/S [DK]. Inventors: Ye Liu [CN], Kirk Matthew Schnorr [DK], Lars Kiemer [DK], Lars Kobberoee Skov [DK], Dorthe Hoej Sandvang [DK], Marianne Thorup Cohn [DK], Ming Li [CN].
