Using Insect Cells For Making Mammalian Proteins

Recombinant protein expression is a major part of biological research. In theory, once the genetic code of a protein is known from cDNA analysis or whole genome sequencing, any polypeptide of interest, existing in nature or perceived, can be artificially produced. Bacteria cells are commonly used to express a variety of proteins because they are more convenient and less costly than other systems. However, a significant percentage of proteins naturally expressed in mammalian cells are not soluble or cannot be easily produced in bacteria such as E. coli. Like bacteria, yeasts are also easy to culture and manipulate, however, although they are eukaryotes, they are not capable of adding “mammalian-like” post-translation modifications (PTM). Insect cells can be used effectively for producing large quantities of mammalian proteins rather easily through baculovirus such as Allele´s Sapphire system. PTM in insect cells is not exactly the same as in mammalian cells, e.g. different glycosylation patterns, but is a lot closer than yeasts. Mammalian cells are used for proteins that require appropriate PTM or are not soluble in other systems through either transient transfection or stable cell line establishment.

For protein expression in insect cells, a number of factors need to be taken into consideration:

1) Genomic DNA for creating baculovirus stocks that will ensure a high percentage of recombinant virus (to avoid wild-type, non-producing virus)
2) Transfer plasmid for cloning the protein-encoding cDNA for easy cloning and appropriate co-expression of helper or marker proteins (such as through insect IRES)
3) Cell lines that have the highest expression levels of a particular protein, sometimes a number of cell lines need to be screened
4) Cell medium, because insect cell medium may contain high levels of ions that can interfere with affinity tag-based purification, one needs to find the most appropriate medium for protein expression
5) Secreted vs nonsecreted proteins. Insect cells need to have their own secretion signal (and translation signal, IRES, polyadinylation, etc.)

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Wednesday, February 29th, 2012 Viruses and cells No Comments

Big Potential in Using Protozoans for Producing Mammalian Proteins

Recombinant protein expression is critical for functionally studying proteins, preparing antigens, providing tissue culture growth supplement, and producing certain therapeutic compounds. Like many molecular biology labs, we have used several heterologous protein expression systems over the last decade including E. coli, yeasts, insect cells and mammalian cells from various species. It is widely accepted that these systems present increasing functional relevance from bacteria to mammalian cells, with accompanying increase in difficulty and cost. The benefits of using cells from higher species are often reflected in post-translational modifications (PTMs), such as glycosylation, phosphorylation, etc.

There is yet another system that could be easy to handle while maintaining mammalian-like PTMs–parasitic protozoan Leishmania tarentolae. L. tarenolae is a unicellular organism, its host is lizard. Even though it’s a vertebrate parasite, this species poses no risk to humans. Amazingly, L. tarenolae individuals can be grown on agar plates for clonal selection or in simple liquid media like E. coli. Their optimal growth temperature is 27C, and they do not require shaking; thus they are suitable for growth in insect cell incubators or even at room temperature. The most important advantage of this system is that oligosaccharide structures of proteins produced in this organism resemble those of mammalian cells much more closely than even insect cells, i. e. the N-glycosylation profile can be basically identical to a biantennary fully galactosylated Man3GlcNAc2core-a-1,6-fucosylated structure found in mammalian cells.

IFrom our first-hand experience, the handling of this species is extremely convenient. While we heavily promote the baculovirus expression system (BVES) for most of our custom protein production projects (we carried out one NIH project for producing human glycosylated cancer antigen proteins using a modified BVES recently), we now believe that there is a good chance that many of the proteins we have been producing could be produced in the protozoan system with potentially better efficiency.

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Wednesday, September 28th, 2011 Viruses and cells No Comments