contact@king-theme.com   +1 123-456-7890
    What Insects Did For Glycoprotein Purification

    What Insects Did For Glycoprotein Purification

    A large number of biotechnologically relevant proteins, including enzymes, hormones, cytokines, clotting factors, monoclonal antibodies are biologically active only in the glycoprotein form (1).

    Glycoproteins – proteins with covalently attached oligosaccharide chains – are not the easiest type of proteins to work with. The oligosaccharide (glycan) part, important for the correct folding and/or biological activity of the purified protein, is attached to the synthesized polypeptide posttranslationally. This means that the conventional purification method such as production and purification in E.coli does not work, because bacteria lack posttranslational mechanisms necessary for the sugars attachment to the polypeptide chain. The expression of glycoproteins without their oligosaccharide counterpart, however, often leads to the protein aggregation and their abnormal targeting if eukaryotic cells were used for the production (2).

    2013-06-04-fall_armyworm_protein_production_2

    Figure1 Fall armyworm – what can it be good for? (Picture from Wikimedia Commons )

    Glycoprotein production in human cell culture, while overcoming the problem of glycoprotein aggregation and incorrect targeting, creates a new challenge – human-derived products are subject to very strict quality control on all production stages, including the serum used for human cell culture. After all, they need to pass the European Medicines Agency and U.S. Food and Drug Administration requirements for the recombinant products.
    Figure 2. Fall armyworm cell line Sf21, one of the most popular insect cell lines for recombinant protein production. (Picture from Wikimedia Commons)
    Insect cell lines and baculoviruses, which do not infect human cells, allow bypassing many of the problems of glycoprotein overexpression. Despite being quite far from humans on the evolutionary tree, many fundamental biological processes, including proteins’ glycosylation, work very well in insects. Insect cell lines (ICL), derived from the butterflies fall armyworm (Spodoptera frugiperda) or Cabbage looper (Trichoplusia ni) in conjunction with baculovirus expression systems allow production of many glycoproteins in a serum-free medium.

    Insect cell lines

    The most commonly used insect cell lines, Sf9 and Sf21 from the fall armyworm, are used to amplify the recombinant virus. An additional advantage of the new generation insect cell lines such as cabbage looper’s High Five™ from Invitrogen is that they grow without CO2 at room temperature, as a semi-adherent monolayer or suspension – it’s much easier to work with, to start a facility from scratch (3). In fact, it’s been proposed to use large scale mammalian cell culture facilities or establish new facilities for vaccines production using insect cell lines (4).

    The vaccine against glycoprotein E of Japanese encephalitis virus has been produced in insect cells and has biochemical and biophysical properties equivalent to the mosquito antigen and causes antibodies formation (5). The other candidates for the insect cells produced vaccines include a vaccine against the main antigen determinants of the famous rabies virus, glycoprotein G.

    The use of insect cell lines for vaccine production is not limited to the glycoproteins, five commercially available vaccines for humans and animals, including CERVARIX® and and PROVENGE® for cervical and prostate cancer, respectively, are already in use and at least 8 others are in different stages of drug development (4).
    If your goals are more ambitious than protein purification and functional analysis, check out this blog article with tips about preparing glycoproteins for crystallization.

    2013-06-04-fall_armyworm_protein_production
    References:

    1. Ghaderi, D., Zhang, M., Hurtado-Ziola, N., & Varki, A. (2012). Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation Biotechnology and Genetic Engineering Reviews, 28 (1), 147-176 DOI: 10.5661/bger-28-147
    2. Vanoni, O., Paganetti, P., & Molinari, M. (2008). Consequences of Individual N-glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE Molecular Biology of the Cell, 19 (10), 4086-4098 DOI: 10.1091/mbc.E08-05-0459
    3. Working with insect cells (Invitrogen manual)
    4. Cox, M. (2012). Recombinant protein vaccines produced in insect cells Vaccine, 30 (10), 1759-1766 DOI:10.1016/j.vaccine.2012.01.016
    5. Kuwahara M, & Konishi E (2010). Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by Spodoptera frugiperda cells for use as vaccine and diagnostic antigens. Clinical and vaccine immunology : CVI, 17 (10), 1560-6 PMID: 20668137