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

Takashi Ohki, Sergey Mikhailenko, Tomomi Arai, Shuya Ishii, Shin'ichi Ishiwata

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 × 106 cells/ml) to that at early exponential phase (1.0 × 106 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 1010 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.

Original languageEnglish
Pages (from-to)351-358
Number of pages8
JournalJournal of Muscle Research and Cell Motility
Volume33
Issue number5
DOIs
Publication statusPublished - 2012 Oct

Fingerprint

Myosin Type V
Baculoviridae
Cell culture
Nutrients
Insects
Actins
Cell Culture Techniques
Food
Sf9 Cells
Lactalbumin
Proteins
Myosins
Infection
Actin Cytoskeleton
Polymerization
Assays
Glucose

Keywords

  • Actin
  • Baculovirus
  • Cell High-density
  • Culture
  • Myosin V Insect
  • Nutrient feeding

ASJC Scopus subject areas

  • Physiology
  • Cell Biology
  • Biochemistry

Cite this

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. / Ohki, Takashi; Mikhailenko, Sergey; Arai, Tomomi; Ishii, Shuya; Ishiwata, Shin'ichi.

In: Journal of Muscle Research and Cell Motility, Vol. 33, No. 5, 10.2012, p. 351-358.

Research output: Contribution to journalArticle

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