The baculovirus/insect cell system has been widely used for recombinant protein production. Since the finding that baculovirus was able to infect hepatocytes in 1995, various attempts to utilize baculovirus as a gene delivery vehicle into mammalian cells have been reported. In this study, we intended to explore the possibility of utilizing a baculovirus/mammalian cell system as a nonlytic, continuous protein production system. A recombinant baculovirus vector carrying enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus immediate-early (CMV-IE) promoter was constructed. This virus was used to infect four common mammalian cell lines, and HeLa was found to yield the highest expression level. Additions of butyrate and valproic acid both enhanced the expression level, but butyrate exhibited a more profound effect. More importantly, HeLa cells were found to be superinfected by baculovirus, a result not observed in the conventional baculovirus/insect cell system. The effects of multiplicity of infection (MOI) and infection timing were also compared. High MOI up to 800 increased the expression in the short term (4 days), but the relatively higher cell death and lower cell density compromised the overall protein yield thereafter. The highest overall expression for a long term was obtained at MOI = 200 when the cells were initially infected at the mid-exponential phase and superinfected with additional baculovirus (MOI = 200) together with a one-time supplement of butyrate. In summary, the strategic infection and feeding enhanced the expression level 9-fold (compared with unsuperinfected culture) and prolonged the duration of expression to 16 days. This study reveals that this baculovirus/mammalian cell system has great potential to become a novel continuous, nonlytic expression system.

The objective of this study is to perform a comprehensive enzyme kinetics analysis in view of validating and consolidating a semimechanistic kinetic model consisting of homogeneous and heterogeneous reactions for enzymatic hydrolysis of lignocellulosic biomass proposed by the U.S. National Renewable Energy Laboratory (Kadam et al., Biotechnol Prog 20(3):698-705, 2004) and its variations proposed in this work. A number of dedicated experiments were carried out under a range of initial conditions (Avicel® versus pretreated barley straw as substrate, different enzyme loadings and different product inhibitors such as glucose, cellobiose and xylose) to test the hydrolysis and product inhibition mechanisms of the model. A nonlinear least squares method was used to identify the model and estimate kinetic parameters based on the experimental data. The suitable mathematical model for industrial application was selected among the proposed models based on statistical information (weighted sum of square errors). The analysis showed that transglycosylation plays a key role at high glucose levels. It also showed that the values of parameters depend on the selected experimental data used for parameter estimation. Therefore, the parameter values are not universal and should be used with caution. The model proposed by Kadam et al. (Biotechnol Prog 20(3):698-705, 2004) failed to predict the hydrolysis phenomena at high glucose levels, but when combined with transglycosylation reaction(s), the prediction of cellulose hydrolysis behaviour over a broad range of substrate concentrations (50-150 g/L) and enzyme loadings (15.8-31.6 and 1-5.9 mg protein/g cellulose for Celluclast and Novozyme 188, respectively) was possible. This is the first study introducing transglycosylation into the semimechanistic model. As long as these type of models are used within the boundary of their validity (substrate type, enzyme source and substrate concentration), they can support process design and technology improvement efforts at pilot and full-scale studies.

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Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- and gene-based therapies. Additionally, baculovirus has emerged as a novel gene therapy vector because of its large cloning capacity for insertion of multiple genes, its minimal cytotoxic effects, and its inability to replicate in mammalian cells. These features have prompted efforts to employ baculovirus vectors carrying mammalian expression cassettes for gene delivery into MSCs. This chapter demonstrates the use of GFP expression to monitor baculovirus-mediated gene transfer into MSCs.