Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing

Abstract

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.

Characterization of enzymatic activity of lysozyme in lysozyme–ovotransferrin complex before and after treatment with trypsin

“…For separation of chicken egg white proteins through non‐denaturing IEF using a previously reported method [14–16, 21], sucrose was added to egg white at a final concentration of 40% (m/v). For separation of lysozyme from egg white by non‐denaturing isoelectric focusing (IEF), IEF was done on gel column (30 mm length × 1.3 mm id) within a glass capillary (45 mm length × 1.3 mm id).…”

“…To characterize the enzymatic activity of lysozyme within lysozyme‐binding proteins, extraction from the solution is required after separation of the native forms of lysozyme‐binding proteins. Lysozyme‐binding proteins were extracted from the solution after the mobilization of proteins, followed by separation using non‐denaturing gel IEF [16]. Although a number of lysozyme‐binding proteins can be separated by IEF, several other such proteins cannot be separated by IEF and were thus eluted as a mixture [16].…”

“…Lysozyme‐binding proteins were extracted from the solution after the mobilization of proteins, followed by separation using non‐denaturing gel IEF [16]. Although a number of lysozyme‐binding proteins can be separated by IEF, several other such proteins cannot be separated by IEF and were thus eluted as a mixture [16]. This is because numerous types of proteins having different molecular masses were present in the acidic region (p I 6 or lower); therefore, it was assumed that multiple proteins were mixed in the eluted fraction from this region.…”

Analysis of the degradation of amyloid beta fibrils after separation via the combination of non‐denaturing agarose electrophoresis and Congo red dye staining

“…The electrode buffer was 0.05 M Tris and 0.38 M glycine (pH 8.3). After electrophoresis, the gels were stained in the solution containing 0.1% v/v CBB R‐250, 50% v/v methanol and 7% v/v acetic acid for 15 min, and destained in the solution comprising 20% v/v methanol and 7% v/v acetic acid for 2 h. The separated proteins on 2DE were compared to those of the protein spots .…”

“…Next, the samples were obtained by collecting the supernatant in each microtube after centrifugation (10 000 × g) at 25°C for 30 min. Moreover, 9 mg sucrose was added to the samples, and they were separated via non‐denaturing two‐dimensional electrophoresis (2‐DE) using a previously reported method . In this technique, proteins are separated by isoelectric focusing (IEF) within a glass capillary followed by electrophoresis in polyacrylamide slab gel.…”