Insect cell expression vectors

Also, special vectors allowing expression in both insect cells and mammalian cell cultures from the same vector (pMamaBac [11] andpBacMam [12]) were described, though the amount required for mammalian transfection with one of these vectors is twofold higher than for insect cells, which makes it applicable only for assessment of suitability for a certain cell culture. 

Philipps, B., Forstner, M. and Mayr, L.M. (2005) A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells. Biotechnology Progress, 21 (3), 708-711. 

Huynh, C.Q. and Zieler, H. (1999) Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines. Journal of Molecular Biology, 288 (1), 13—20. 

Insect cells in culture are also hosts for recombinant protein production. Production of recombinant proteins in the baculovirus expression vector system is the most common system. Titers of recombinant protein as high as 11 g/L have been obtained. 

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. 

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). 

Optimal conditions for insect cell growth have been extensively studied, but for product expression with a baculovirus infected insect cell the focus should be on the difference in the metabolic requirements of infected vs. uninfected cells, which has been observed to differ after infection. The alanine specific production rate decreases almost four-fold, while phenylalanine specific consumption rate increase 11-fold and glutamine specific consumption decrease [65]. Both an increase [66] and a decrease [67] in glucose consumption rates of insect cells after infection have been reported. This reflects some differences in the media and vectors that were used however, it is normal to expect a higher metabolic burden after infection due to the increase in protein expression rates caused by the infection. This creates a concern about the impact of nutrient limitations on the productivity of the system. 

The Transdirect insect cell is a newly developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Spodoptera fru iperda 21 (S 21) insect cells. An expression vector, pTDl, which includes a 5 -imtranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of about 50 pg per mL of the translation reaction mixture. This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts. 

The Transdirect insect cell kit is an in vitro translation system for mRNA templates. We developed and optimized a method to prepare the insect cell extract, the concentrations of the reaction components, and an expression vector pTDl (2, 5). The pTDl... 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Suzuki, T., Ito, M., Ezure, T., Kobayashi, S., Shikata, M., Tanimizu, K, and Nishimura, O. (2006) Performance of expression vector, pTDl, in insect cell-free translation system. J. Biosci. Bioen0. 102, 69-71. 

Smith, G.E., Ju, G., Ericson, B.L., Moschera, J., Lahm, H.W., Chizzonite, R. Summers, M.D. (1985). Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proceedings of the National Academy of Sciences (USA) 82, 8404-8. 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

Other protocols involve cell lines with integrated rep/cap cassettes (Clark et al., 1995 Gao et al., 1998 Liu et al., 1999 Chadeuf et al., 2000 Mathews et al., 2002 Qiao et al., 2002a,b) infected with adenovirus or, alternatively, a recombinant herpesvirus system has been used to provide both helper virus function and rep/cap (Conway et al., 1997, 1999). In a switch away from using mammalian cell and helper virus production systems, rAAV vectors have been made in insect cells where the AAV genes are expressed under the control of insect promoters and the traditional helper virus gene products are not required (Urabe et al., 2002). Stable producer cell... 

It is worth emphasizing that all biopharmaceuticals mentioned here are produced from mammalian cell culture. The protein production system based on insect cells known as BEVS (baculovirus expression vector system) is widely employed for the expression of a wide range of proteins, but, due to regulatory issues, biopharmaceuticals produced by insect cells are not yet in the market. However, some of them are being evaluated,... 

The expression systems available for the production of recombinant proteins in animal cells can be classified as mammalian cell/viral or plasmid vector, or insect cell/baculovirus. 

Farrell PJ, Lu M, Prevost J, Brown C, Behie L, Iatrou K (1998), High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector, Biotechnol. Bioeng. 60 656-663. 

Jarvis DL, Finn E (1996), Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors, Nat. Biotechnol. 14 1288-1292. 

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