Insect cells infected

Smith, G. E., Summers, M. D., and Fraser, M. J. (1983). Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol. Cell Biol. 3, 2156-2165. 

The DDI fluorescent inhibition screen is a high-throughput screen, primarily as a result of reduced analysis time, and can range from a few hundred compounds per week to a thousand per week. The assay uses recombinant microsomes prepared from insect cells infected with recombinant baculovirus containing a human CYP enzyme and individual fluorogenic substrates. In this instance the substrates are not specific and hence cannot be used in a mixed enzyme system such as HLM. In addition, this assay is more prone than the conventional inhibition screen (Section 8.3.2) to NCE interferences within the assay (i.e., inherently fluorescent NCEs, fluorescent quenching by the NCE). 

Two different protein phosphatases were used the one from Upstate Biotechnology (New York, USA), from human red blood cells, and the one from GTP Technology (Toulouse, France), isolated from SF9 insect cells infected by baculovirus. The enzymatic activity of these two enzymes towards several substrates was investigated by cyclic voltammetry and steady-state chronoamperometry (see experimental details in Refs. [86,87]). First, commercial substrates were tested. Ascorbic acid 2-phosphate and phenyl phosphate were not recognised by the protein... 

Expression of viral structural proteins in a heterologous system does not always lead to formation of the desired assembly intermediate or end product. This is particularly the case when the proteins are expressed in E. coli or when individual components are expressed separately in different cells. In these cases, the proteins are usually purified and then assembled in vitro. Alternatively, assembly is possible using whole cell lysates. For example, assembly of HSV-1 capsids, which requires a minimum of four structural proteins, was observed on mixing of lysates derived from insect cells infected with different baculovirus vectors (Newcomb et al, 1994). Similarly, reovirus cores, obtained from native virions, could be... 

Trus, B. L., Homa, F. L., Booy, F. P., Newcomb, W. W., Thomsen, D. R., Cheng, N., Brown, J. C., and Steven, A. C. (1995). Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses Structural authenticity and localization of VP26./. Virol. 69, 7362-7366. 

Lee CA, Kadwell SH, Kost TA, Serabjit-Singh CJ. CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalyticaUy similar to human liver microsomal CYP3 A4. Arch Biochem Biophys 1995 319 157-167. 

Since the exact titer of stock viruses is not determined in this simplified protocol, it is recommended that three different dilutions of viruses are tested (1 50, 1 100, 1 200) for insect cell infection for protein expression. 

Insect Cells. In this system the cDNA is inserted into the genome of an insect vims, baculovims. Insect cells, or Hve insect larvae, are then infected with the vims. In this way advantage is taken of the vims s natural machinery for repHcation utilizing the insect cell. This is one of the best systems available for high level production of native protein having post-translational modifications similar to those seen in mammalian cells. Disadvantages of this system include lytic—batch variations, comparatively slow growth, and cosdy scale-up. 

A number of allergens from both honey bee and vespid venoms have been cloned and expressed by either Escherichia coli or baculovirus-infected insect cells (table 1) phospholipase Aj [20], hyaluronidase [21], acid phosphatase [13] and Api m6 [14] from honey bee venom, as well as antigen 5 [22], phospholipase A and hyaluronidase [23] from vespid venom, and dipeptidylpeptidases from both bee and Vespula venoms [15, 16]. Their reactivity with human-specific IgE antibodies to the respective allergens has been documented [11-16, 22, 23] and their specificity is superior... 

A wide range of proteins have been produced at laboratory scale in recombinant insect cell culture systems. The approach generally entails the infection of cultured insect cells with an engineered baculovirus (viral family that naturally infect insects) carrying the gene coding for the desired protein placed under the influence of a powerful viral promoter. Amongst the systems most commonly employed are ... 

Human pathogens (e.g. HIV) do not generally infect insect cell lines. 

Aside from the cysteine motif, conserved regions in the proteins encoded by WHnl.O and WHul.6 are likely to reflect functional roles. Based on the rules of Von Heijne (70), the N-terminal 16 amino acids may function as a signal peptide for secretion. Recombinant WHbl.O and WHt/1.6 proteins are secreted into the medium of infected insect cells (43)... 

Baculovirus Improved procedure Infection of insect cells High expression yields Relatively slow virus production Different post-translational processing... 

Post-translational modifications, such as phosphorylation, complex glycosylation, and lipidation, typically occur in eukaryotic organisms. Therefore, their expression in prokaryotic systems like Escherichia coli is difficult. However, it should be noted that via clever engineering and coexpression of specific enzymes, access can be granted to specific lipidated proteins via expression in bacteria, for example, via the expression of A -myristoyltransferase in E. coli Eukaryotic systems that can be used for the expression of post-translationally modified proteins are yeast and Dictyostelium discoidum. Furthermore, lipidated proteins, such as the Rah proteins, can be obtained via purification from tissue sources or from membrane fractions of insect cells that had been infected with baculovirus bearing a Rah gene. ... 

Baculovirus expression is the most frequently used method for expression in insect cells and employs Autographa californica nuclear polyhedrosis virus (AcNPV), a double stranded (ds) DNA virus that infects arthropods. The baculovirus expression system utilizes features of the viral life cycle to introduce recombinant DNA coding the gene of interest into insect cells (Miller, 1988 O Reilly et al, 1992). 

Production of Core and Virus-Like Particles with Baculovirus Infected Insect Cells... 

In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed. The issues addressed include particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process. Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes. In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters. Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression. 

Viral structural proteins expressed by baculovirus infected insect cells assemble into multimeric structures that resemble viral core-like particles and virus-like particles (CLPs and VLPs, respectively). This presentation has brought the attention of researchers for the potential use of these structures as safe immunological reagents for virus or antibody detection in enzyme immuno assays, as vaccines, and more recently, as gene delivering systems for gene therapy [9]. 

In this paper the fundamental aspects concerning the production of CLPs and VLPs with baculovirus infected insect cells are reviewed. It is not the goal of this communication to review all the aspects of insect cell culture technology, since this would be a task impossible to achieve in a single chapter. The interested reader should refer to the references. This review is structured in four parts ... 

In this part we deal with the major chemical and physical factors affecting insect cell growth, baculovirus replication and product expression. The issues addressed are cell line selection for product expression, baculovirus-cell interactions and impact of differences in metabolic requirements of infected and non-infected cells in overall productivity. 

In this part the application of mathematical models to CLP and VLP production with baculovirus infected insect cell cultures is discussed. Special emphasis on model evaluation is made along with the definition of directions in future process development research with this system. 

For the production of CLPs and VLPs with baculovirus infected insect cells the specific proteins that are required for particle formation should be chosen at an early step. The specific particle composition, along with the expression of other non-structural (NS) proteins often has a great impact on particle stability [11] or on particle localisation [12], i.e. - cell-associated or secreted to the supernatant. 

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