title: Kinetics of infected insect cell osmolysis and enhanced protein release using a modified disruption method.
abstract: We have studied and characterized a cell disruption method to produce a protein extract from recombinant Baculovirus infected insect cells based on osmotic lysis. Cell lysis kinetics were measured during a 24-h incubation in lysis buffer and resulting data sets were curve fitted to a hyperbola, visually similar to the Michaelis-Menten curve, to determine the maximum concentration of released protein and the time required to reach equilibrium. Effect of parameters such as pH, ionic strength and infection phase were evaluated, and based on fittings optimal protein release conditions were obtained for total cell protein as well as the recombinant protein, HPV 16 L1. It was demonstrated that pH and the phase of infection can vastly influence the amount of release while ionic strength only effects the time required to achieve equilibrium in protein release. Osmolysis can be a mild, yet effective method to release recombinant protein with high recovery levels and hence can be used in capacities where stringent criteria regarding contamination with surfactant or non-cytoplasmic contents are observed.
authors.affiliation: Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran.
authors.forename: Reihaneh
authors.initials: R
authors.lastname: Mirhassani
authors.affiliation: Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran. seyedjafari@ut.ac.ir.
authors.forename: Ehsan
authors.initials: E
authors.lastname: Seyedjafari
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D036022:Capsid Proteins; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; C063317:L1 protein, Human papillomavirus type 16
keywords: Cytolysis; HPV; Hypotonic lysis buffer; Insect-baculovirus