Lysozyme purification

Mixture of proteins (Myoglobin, Ovalbumin, Lysozyme and Chymotrypsinogen) Purification Hydrophobic Interaction [52]... 

A separation of myoglobin and lysozyme has been presented by Nicoud [44]. This purification was performed on SMB containing 8 columns using ACA 54 (Biosepra, France) as support. Very pure extracts (>98%) and raffinates (>98%) were obtained from a 50-50 mixture. An internal profile is given in Fig. 7. Another recently presented example is the separation of cyclosporine A from cyclic oligopeptide and other impurities in the reversed phase mode or by adsorption on silica gel presented by Schulte et al. [10]. 

M.Y. Arica, M. Yilmaz, E. Yalcin and G. Bayramoglu, Affinity membrane chromatography relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification. J. Chromatogr.B, 805 (2004) 315-323. 

The harvested cells were dispersed in 100 mL of purification buffer A containing 50 mM sodium acetate (pH 5.0), 2 mMDTT, and then disrupted using lOmgofhen egg lysozyme and 15 min of sonication. 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

Thomas, B. R., et al.. Distribution coefficients of protein impurities in ferritin and lysozyme crystals - Self-purification in microgravity. J. Cryst. Growth 2000,... 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

The classical column procedures for the preparation of lysozyme are difficult to use with high viscosity solutions such as serum. A batch method was developed based on affinity chromatography using deami-nated chitin [82]. The method was found to give a one-step purification of nearly theoretical amounts in tissue homogenates tested. These included tissue homogenates from humans, primates, avian egg white and plants. The use of a chitin-coated cellulose affinity column for purification of lysozyme has also been described [83]. 

Angiotensin-I, methionine enkephalin, substance-P, bovine trypsin, horse cytochrome-C, horse myoglobin, bovine insulin and egg-white lysozyme were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. 

Table 4. Purification factors for the recovery of lysozyme from a 1 1 ovalbumin-lysozyme solution by precipitation with CMC at pH 4.2 and 0.02...

Ionic Strength Dosage [M] Protein removals Lysozyme Ovalbumin Purification factor Purity of lysozyme in solid... 

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