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.

Protein production using the baculovirus‐insect cell expression system

“…Batch and fed‐batch cultures are the preferred modes of operation for large‐scale production of proteins via the BEVS . This is because batch and fed‐batch operations are simple, flexible, and inherently suited to a process involving a lytic infection cycle.…”

“…In perfusion culture, the bioreactor is fed continuously and the harvest is continuous at the same volume flow rate as the feed rate, but the cells are retained within the bioreactor by some sort of retention mechanism. Although a perfusion culture is more complex than a fed‐batch operation, it is being increasingly used . In a perfusion operation with a possible periodic bleed of cells, the nutrient levels can be maintained at steady values and the product protein is continuously removed in the spent medium leaving the bioreactor.…”

A re-usable wave bioreactor for protein production in insect cells

“…Most common are bioreactors especially in the pharmaceutical industry, albeit less represented in academic laboratories. In the past few years, alternative choices have emerged, like high cell density protocols [4] or culture vessels that can be filled up to 5 L [5] . Having Labfors stirred-tank bioreactors and Wave Platforms available, we invested efforts in bioreactor cultivation.…”

Polymer-antibody fragment conjugates for biomedical applications

“…1), such as hexahistidine motifs, to the N-or C-terminus of the protein is often carried out during cloning to facilitate detection and purification of the expressed protein using generic protocolsand equipment while the tag can be engineered for removal after protein purification. Other host systems such as yeast [38], insect [39] or mammalian [40] cells also offer specific advantages such as the ability to introduce post-translational modifications lacking in E. coli or improved expression of multi-subunit or complex proteins (reviewed in [35]). …”