title: Improvement of the yields of recombinant actin and myosin V-HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture.
abstract: Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V-HMM at late exponential phase (11.3 × 10(6) cells/ml) to that at early exponential phase (1.0 × 10(6) cells/ml). The relative yields of actin and myosin V-HMM were approximately equal at both phases (typically 200 mg of actin and 5 mg of myosin V-HMM per 10(10) cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V-HMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay.
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Tokyo 169-8555, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohki
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Mikhailenko
authors.forename: Tomomi
authors.initials: T
authors.lastname: Arai
authors.forename: Shuya
authors.initials: S
authors.lastname: Ishii
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Ishiwata
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000096:biosynthesis*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D024701:Myosin Type V / Q000096:biosynthesis*; D009218:Myosins / Q000032:analysis; D058105:Polymerization; D011994:Recombinant Proteins / Q000096:biosynthesis*; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D024701:Myosin Type V; D009218:Myosins