Factors influencing recombinant human lysozyme extraction and cation exchange adsorption.
Human lysozyme has numerous potential therapeutic applications to a broad spectrum of human diseases. This glycosidic enzyme is present in tears, saliva, nasal secretions, and milk--sources not amendable for commercial development. Recently, a high expression level of recombinant human lysozyme (0.5% dry weight) was achieved in transgenic rice seed. This paper evaluates the effects of pH and ionic strength on rice protein and lysozyme extractability, as well as their interactions with the strong cation-exchange resin, SP-Sepharose FF. The extraction conditions that maximized lysozyme yield and the ratio of extracted human lysozyme to native rice protein were not optimal for lysozyme adsorption. The conditions that gave the highest extracted lysozyme to native protein ratio were pH 4.5 and 100 mM NaCl in 50 mM sodium acetate buffer. At pH 4.5, salt concentrations above 100 mM NaCl reduced the lysozyme-to-protein ratio. The best conditions for lysozyme adsorption were pH 4.5 and 50 mM sodium acetate buffer. Lysozyme extraction and subsequent adsorption at pH 4.5 and 50 mM NaCl was an acceptable compromise between lysozyme extractability, adsorption, and purity. The primary recovery of human lysozyme from pH 6 extracts, irrespective of ionic strength, was inferior to that using pH 4.5 with unacceptably low saturation capacities and lysozyme purity. High purity was achieved with a single chromatography step by adjusting the pH 4.5 extract to pH 6 before adsorption. The disadvantage of this approach was the drastically lower saturation capacity compared to adsorption at pH 4.5.
Wilken
Lisa R
LR
Biological and Agricultural Engineering, Texas A&M University, 2117 TAMU, College Station, Texas 77843, USA.
Nikolov
Zivko L
ZL
D000327:Adsorption; D001665:Binding Sites; D002411:Cation Exchange Resins / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry / Q000378:metabolism; D019346:Sodium Acetate / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry
D002411:Cation Exchange Resins; D002412:Cations; D011994:Recombinant Proteins; D012965:Sodium Chloride; D019346:Sodium Acetate; D009113:Muramidase