Insect cell expression vectors system

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. 

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. 

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. 

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 values of qp can vary significantly as a function of the expression system used and of the recombinant protein product. In general, values in the range of 1 —30 pg cell-1 day-1 are reported for stably transfected cell lines. However, Chico and Jager (2000) have reported values as high as 85 pg cell-1 day-1 for the expression of a protein using the insect cell-baculovirus expression vector system. This value approaches the maximum theoretical value that is expected for animal cells, which is 100 pg cell-1 day-1 (Ozturk, 1990). 

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). 

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... 

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. 

Recombinant baculoviruses have been used as vectors to express heterologous proteins in cultured insect cells (3). A number of unique features distinguish the baculovirus expression system from other expression systems ... 

Numerous shuttle vectors are available that facilitate convenient transfer from bacteria or mammalian cells. Proteins such as HIV Gp 160, carcinoembryonic antigen, and a form of influenza vaccine have successfully been expressed in baculovirus systems [9]. Potential disadvantages of this system include inappropriate posttranslational modifications, use of insect rather than mammalian cells, and characterization of both the baculovirus and SF9 insect cells used. 

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