Licari, Peter (1992) An engineering analysis of the insect cell - baculovirus expression system. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08072007-103816
Baculovirus expression vectors are routinely used for the expression of heterologous proteins, affording the user relatively high product yields and a variety of post-translational modifications. The insect cell-baculovirus system utilizing the Autographa californica multicapsid nuclear polyhedrosis virus has been studied here with regard to product yield and product quality. Product yield was demonstrated to be affected by both the time of infection and the multiplicity of infection. Through experimental results and population modeling of infected cultures, the product yield was maximized by infecting in the early-exponential growth phase with a low multiplicity of infection.
The importance of intracellular protein degradation on cloned protein yield and product quality in this system was also studied. Intracellular degradation was observed throughout the infection; however, when compared to the synthesis rate, degradation was found to have an insignificant effect on system productivity. A population of discrete [beta]-galactosidase fragments was observed in protein-radiolabelling experiments. The synthesis rate of these fragments compared to the degradation rate of the complete [beta]-galactosidase molecule suggested that they are probably not a result of intracellular protein degradation. Although proteolytic origin cannot be excluded, transcript hybridization analysis suggests that premature transcript termination may be the cause. It is believed that after premature transcription termination, the transcript is translated to yield the fragmented polypeptides. Other evidence using different recombinant viruses, all expressing related forms of [beta]-galactosidase, are consistent with the hypothesis of premature transcription termination. Although the synthesis of [beta]-galactosidase was most thoroughly studied, transcript heterogeneity and protein heterogeneity were observed for a variety of recombinant viruses with the heterologous gene under the control of the polyhedrin promoter.
When expressing a heterologous glycoprotein, the insect cell-baculovirus expression system produces a heterogeneous population of glycoforms different from those of authentic mammalian hosts. Four different cell lines that can be used as hosts for baculovirus expression were analyzed for the existence of endogenous exoglycosidases. Cells derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, and Malacosoma disstria contained N-acetyl-[beta]-glucosaminidase, -acetyl-[beta]-galactosaminidase, [beta]-galactosidase, and sialidase activities. These endogenous activities were also observed in the medium from uninfected and wild-type baculovirus-infected cultures. Lectin analysis of insect cell glycoproteins indicates that oligosaccharides formed in these hosts are susceptible to the exoglycosidase activities identified.
On the basis of reports that insect cells lack terminal glycosyltransferases, mammalian [beta]1,4-galactosyltransferase and [alpha]2,6-sialyltransferase were cloned into baculovirus expression vectors. The degree of altered glycosylation resulting from the expression of these enzymes in insect cells, alone or together, will require more sensitive carbohydrate analysis than that provided by lectins.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Chemical Engineering|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Awards:||Constantin G. Economou Memorial Prize, 1990|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||1 May 1992|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||10 Aug 2007|
|Last Modified:||11 Dec 2014 00:11|
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