glycosylation

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

Making Recombinant Glycosylated Proteins (I)

Many mammalian cell membrane-bound and secreted proteins are glycosylated. The degree of their modifications may be dependent upon tissue specificity and cellular states such as normal vases cancerous. The existence of these proteins in bodily fluids, in combination to their relevance to diseases, makes glycosylated proteins good candidates for clinical diagnostics. Understanding the biogenesis, structure, and functions of these proteins will also aid research and prevention of cancers.

Cancer formation is a heterogeneous and complex process, involving many factors and cellular signaling pathways in each type of cancer. There are more than 1,200 potential cancer biomarkers identified in the literature by a 2006 review. We found that ~ 70% of the 1,261 proteins listed in are naturally secreted proteins, and some 40-50% glycosylated. The addition of carbohydrate groups during protein glycosylation to asparagines (N-linked), or threonines or serines (O-linked) residues may result in mono-, disaccharide- or branched oligosaccharide composed of as many as 20 monosaccharide residues. Glycosylation, together with other modifications, often change the apparent molecular mass of a secreted protein to many folds to that predicted by amino acid sequence. Such heavy modifications on the surface of proteins can influence their functions as well as characteristics as antigens or analytes. Studies of glycosylated proteins offer great opportunities for improving cancer diagnostics.

There are increasing demands for these glycosylated human proteins in good quantity, purity and affordability by the scientific community to perform fundamental and clinical studies in relation to cancer. Such proteins cannot be expressed in bacteria or yeast because those cells do not carry out equivalent post-translation modifications (PTM) as in mammalian cells. Although there have been successful attempts to modify yeast cells to produce proteins with certain types of glycans attached, they were designed for expressing a few pharmaceutical proteins and not suitable for expressing a wide variety of cancer markers. Aside from PTM, expressing human proteins in microorganisms may be hindered by their different codon usage preferences and protein folding tendencies.

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    Wednesday, January 26th, 2011 Uncategorized No Comments