Competition For Cellular Resources Research Articles

Improved expression of secreted and membrane-targeted proteins in insect cells

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.

Multiplicity and stability of steady states in continuous bioreactors: dissection of cybernetic models

Microbes in industrial bioreactors generally encounter nutrient media that contain a spectrum of components satisfying various nutritional requirements with individual substrates playing roles that may be viewed as substitutable, complementary or combinations of the two to varying extents. Consequently, the pattern of uptake of different nutrients (Pavlou and Fredrickson, Biotechnol. Bioengng 34(7) (1989) 971) by the organism is linked through metabolic regulation of its biochemical pathway to its environment as determined by the composition of the surrounding nutrient medium. Such a situation is indicative of an abundance of steady state multiplicity in continuous bioreactors where different steady states may feature cells with notably different physiological states and consequently vastly different metabolic activities. A striking example of such steady state multiplicity is that observed by Hu et al. (J. Microbiol. Biotechnol. 8(1) 8), and Follstad et al. (Biotechnol. Bioengng 63(6) (1999) 675) with hybridoma cell cultures for the production of antibodies. A mathematical investigation of the foregoing steady state multiplicity must obviously involve models that incorporate metabolic regulation. In this regard, the cybernetic models developed by Ramkrishna and coworkers (Kompala et al., Biotechnol. Bioengng 28 (1986) 1044; Baloo and Ramkrishna, Biotechnol. Bioengng 38(11) (1991a,b) 1337, 1353; Straight and Ramkrishna, Biotechnol. Prog. 10(6) (1994) 574; Ramakrishna et al., Biotechnol. Bioengng 52(1) (1996) 141) represent an ideal framework of which to initiate such an investigation. These models account for regulation on suitably simplified pathways by viewing it as the competition for cellular resources between different enzyme systems. Such competition may produce outright victors denoted by zero enzyme levels of those vanquished or simultaneous expression and activation of all the competing enzyme systems. The cybernetic models feature the foregoing competition with cybernetic variables u i for the regulation of synthesis of the ith enzyme, and v i for its activation. The latter cybernetic variables are not differentiable at points where reversal of competition occurs so that the application of nonlinear bifurcation analysis based on calculation of the Jacobian must take due account of this technical difficulty. The strategy proposed here for the bifurcation analysis of cybernetic models is based on a combinatoric approach enumerating all possible consequences of competition between different enzyme systems. Since the system for each combinatoric is differentiable, a bifurcation analysis is therefore enabled. Furthermore, a notable feature is that the procedure analytically reduces the system to one of a single dimension by purely analytical means. The bifurcation analysis is performed on the model of Kompala et al. (Kompala et al., 1986), as well as that of Ramakrishna et al. (Ramakrishna et al., 1996). The latter is a refinement of the former, which describes sequential as well as simultaneous utilization of multiple substrates. The results of our analysis on both models demonstrate the existence of multiple, stable, steady states for certain ranges of our bifurcation parameters, D the dilution rate and γ the fraction of the more preferred substrate in the feed. These multiple steady states refer to different levels of utilization of the more preferred substrate. This multiplicity occurs for the model of Kompala et al. (Kompala et al., 1986) at D near the maximum dilution rate and γ suitably small. However, multiplicity exists for the model of Ramakrishna et al. (Ramakrishna et al., 1996) even when the preferred substrate is in excess. The reasons are discussed. The methodology has potential for considerably more detailed cybernetic models and hence should be of practical significance to biotechnology.