Mammalian and Insect cells

The utilization of mammahan and insect cell cultures for the expression of heterologous proteins, such as hormones, enzymes, cytokines, and antibodies, for research or human therapy has steadily increased over the last several decades [104, 105]. In comparison with microorganisms and plant cells, mam-mahan/insect ceUs are the preferred expression system for complex human proteins because of their abihty to synthesize and secrete a wide variety of functional authentic proteins with proper folding/assembly and human-like complex glycan structures [106]. 

Common features of mammalian/insect cells include slow grorvth, relative fragihty, complex metabohsm, complex medium requirements, and possibly. 

Products Plant species Immobilized Fold/percentage References 

Secondary metabolites Alizarin and Rubia tinctorum L. Entrapment in loofa, [94] 

Diosgenin Dioscorea deltoidea Entrapment in polyurethane foam Increase by 40% [96] 

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Lohse J, Dahl O, Neilsen PE (1999) Double duplex invasion by peptide nucleic acid a general principle for sequence-specific targeting of double stranded DNA. Proc Natl Acad Sci USA 96 11804-11808 Lonn U, Lonn S, Nylen U, Windblad G (1990) Bleomycin-induced DNA lesions are dependent on nucleosome repeat length. Biochem Pharmacol 39(1) 101-107 Lopez-Larraza DM, Bianchi NO (1993) DNA response to bleomycin in mammalian cells with variable degrees of chromatin condensation. Environ Mol Mutagen 21(3) 258—264 Lopez-Larraza DM, Padron J, Rond NE, Vidal Rioja LA (2006) Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin. Mutat Res 16 April [Epub ahead of print]... 

Various strategies have been used to combine the variable region of antibodies, which bind to the antigen determinants, with small functional proteins. Such constructs can be produced on a large scale in various expression systems (Irving et al., 1996 Roque et al., 2004) bacterial expression systems are relatively simple and less expensive than the alternatives, but eukaryote expression systems (yeast, mammalian, and insect cells) are also being used for this purpose (Roque et al., 2004). 

Ma, N., K.W. Koelling, and J.J. Chalmers. 2002. Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces. Biotechnol Bioeng 80 428-437. 

Tramper J Vlak JM (1988) Bioreactor design for growth of shear-sensitive mammalian and insect cells. In Mizrahi A (ed.) Advances in Biotechnological Processes, vol. 1, pp. 199-228. Alan R. Liss, New York. 

Site-directed mutagenesis has been done using mammalian and insect cell-based systems, and models have been in existence for some time. More recent modeling work - has been done, with an emphasis on docking of inhibitors in SRS-1. 

Techniques of attachment, entrapment, and encapsulation are most widely used for cell immobilization with support materials, which are illustrated in Figure 7.1. These techniques can be applied to essentially all the viable or nonviable wholecell systems of potential interest microorganisms, plant cells, and mammalian and insect cells [2]. Although most of the principles associated with enzyme immobilization are directly applicable to cell immobilization, due to the complete difference in size and biochemical properties between enzymes the cells, the relative importance of these methods is considerably different [10]. 

Immobilized Cells and their Applications 223 Table 7.3 Recombinant proteins produced by immobilized mammalian and insect cells. 

Keeton, T.P. and L.A. Bulla, Jr. 1997. Ligand Specificity and Affinity of BT-Rl, the Bacillus thuringiensis CrylA Toxin Receptor from Manduca Sexta, Expressed in Mammalian and Insect Cell Cultures. Appl. Environ. Microbiol. 63 3419-3425. 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Standard temperatures are usually defined for every known cell line, for example, mammalian cell lines are usually cultured at 37°C and insect cell lines at 28°C. However, studies have demonstrated that temperature modifications can lead to changes in certain parameters, such as increased productivity of recombinant proteins or decreased byproduct generation. 

Animal cell cultures, either from mammalian or insect cells, show specific characteristics that differ from microbial cultures (bacteria, yeasts, and filamentous molds). Among the characteristics that most interfere with monitoring methodologies and process control (and that will be approached with more detail in the present chapter), are the following. 

Most of the expression vectors currently in use are constructed and propagated in E. coli bacterium. Large amounts of the vector DNAs are extracted from E. coli cultures and introduced into the chosen host cell. (A notable exception to this generalization are vectors derived from mammalian and insect viruses, but even in these cases at least some of the components of the expression vector are assembled in E. coli.) Thus, vectors assembled by the manipulation of DNA by restriction nucleases and ligase are introduced in the naked-DNA form into E. coli rendered competent to accept extraneous DNA. This process is termed Transformation Once the vector enters the E. coli cell, it has to be replicated and maintained in order to avoid its loss due to the dilution that accompanies rounds of cell division. The presence of a DNA sequence on the vector which functions as the origin of replication permits the vector to replicate in the host cell. 

Fig. 5 The single-cell analysis chip and the analysis of P2AR in Sf9 cells. A, Layout of the single-cell chip, showing the cell-manipulation section on the left and the molecule-counting section on the right B, Analysis procedure for a mammalian or insect cell ...

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