Team:Tec-Monterrey/allergen/project

P.pastoris is transformed with the necessary sequences for the production and secretion of the three allergens and two single chain variable fragments. Proteins must be purified y affinity chromatography and concentrated by centrifugal filtration. Finally, our kit is assembled and it's ready for an allergy detection assay, which only needs bloods serum and gives GFP emission as a positive response.

The presence of allergic individuals in the population has been increasing and becoming more common. The key to preventing allergic conditions is screening and early detection of reactions to different allergens. Synthetic biology can be used as a new approach to develop a standardized production of allergen proteins and most of the components needed to produce an allergy detection kit. The main expression system that we used to produce the necessary proteins for this project was Pichia pastoris. Yeasts like P. pastoris are valuable to synthetic biology for their folding and secreting capabilities4. Also, as an eukaryote, P. pastoris can produce proteins with all the post-translational modifications which are essential for their activity5. We designed a functional novel shuttle expression sequence for the expression of proteins in both P.pastoris and E.coli, by doing so, scientists can harvest the benefits from both expression systems while maintaining the same coding sequence.

Our project can be easily explained in 3 images. First, we have to transform our yeast strains with the different proteins for the allergen detection kit, coded in a linearized plasmid. The internal transcription, translation, and secretion mechanisms in Pichia pastoris will start making and exporting our protein out of the cell. Finally, after a purification process for the proteins, they can be used for an allergy detection kit, giving off a green fluorescence as a positive result.

The presence of allergic individuals in the population has been increasing and becoming more common.^[2] In recent years, allergic diseases have had a significant increase in Latin America, becoming a public health problem in many countries.^[3] The concept of "allergy" was introduced in 1906 by Austrian pediatrician Clemens von Pirquet, after he noticed that some of his patients were hypersensitive to normal harmless substances such as dirt, pollen, or certain foods. Pirquet called this phenomenon "allergy" from the Greek allos meaning "other" and ergon meaning "work"^[6]. He recognized individuals with hypersensitivity to certain compounds when an antigen induced "altered reactivity".^[4]

Presenting allergies to environmental agents can affect any organ of the body.^[2] These agents are called "allergens" which are substances (usually proteins) that may indicate a hypersensitivity to the person with whom it had contact with1. Typical allergens allergens are dust mites, pollen from trees, grasses and molds, cats, insects like wasps and bees, milk, eggs and nuts. Least common ones include certain fruits and latex. There are other non-proteic allergens, including penicillin and other drugs ^[1].

In Latin America, one of the most common allergic conditions is allergic rhinitis. In Mexico, 40% of children between 13 and 14 years old suffer from it.^[3] The majority of the scientific field is focused in the diagnosis and treatment of stationary allergic conditions such as rhinitis, allergic asthma, insect allergies, anaphylaxis and food intolerance as well as skin disorders such as hives, angioedema or atopic eczema.4 It is clear that allergic diseases are related to genetic heritage but it has been difficult to conduct genetic studies because of the many different markers and allergic diseases that exist.^[4]

At the time of birth, the immune system determines to be prone to allergies (TH2) or not-prone to allergies (TH1) depending on genetics and the environment. TH1 immunity is efficient protecting against bacteria and viruses, as well as allergies. The immunity TH2 is effective against parasitic infections but makes the individual very vulnerable to develop allergies. If an individual has a family history of allergic conditions is much more likely to develop type TH2 immunity which promotes the production of large amounts of immunoglobulin E, an allergy-related immunoglobulin in the blood.^[5] Other factors that may influence whether TH1 or TH2 cells dominate the response to an allergen; include the allergen dose and exposure time as well as the ability of the allergen to interact with cells.^[4]

The discovery of Immunoglobulin E (IgE) antibody by Kimishige Ishizaka and colleagues was an important breakout in understanding the mechanisms of allergies. They were the first to isolate and describe IgE in the 60's.^[3] The immune system of allergic individuals produces the IgE antibody as a response when it recognizes substances as harmful allergens. The IgE orders other blood cells (mast cells) to produce chemicals (such as histamine) that cause irritation, inflammation and the symptoms of an allergic response.^[1] Acute allergic reactions result in the release of mediators such as membrane lipid derivatives like cytokines and chemokines when an allergen interacts with the IgE attached to the mast cells.^[4]

According to experts, the key to preventing allergic conditions is screening and early detection of reactions to different allergens. But although some diagnosis are relatively simple, less obvious symptoms can be presented that require extreme vigilance.^[3] The diagnosis of allergens or triggers of allergic diseases is essential for the generation of appropriate methods to avoid, treat, and control environmental factors around the affected individual.^[2]

Pichia pastoris a new member in synthetic biology

As a unicellular eukaryote, yeast can potentially produce soluble, correctly folded recombinant proteins that have undergone all the post-translational modifications that are essential for their functions .The organism is quite safe thanks to the absence of endotoxins and oncogenes. Yeast cells are also easier to culture and manipulate genetically than mammalian cells and can be grown to high cell densities, around 50% w/v. (Yin Et al., 2007)

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. Up to 2000, more than 200 proteins from viruses, bacteria, fungus, animals, plant and human beings have successfully been expressed in P. pastoris. Evidently, the use of a shuttle vector facilitates the transformation of yeast. Shuttle vectors which can replicate in two different host species (E. coli and yeast) were used to transform foreign gene into yeast cells and the transformations consist of replicative and integrative transformations. A further benefit is that strong promoters are available to drive the expression of a foreign gene of interest, facilitating the production of large amounts of target protein more inexpensively than most other eukaryotic systems. Nevertheless, this “lower” eukaryote differs from its mammalian counterparts in the way it forms both Nand O-linked oligosaccharide structures on target. Recently, advances in the glycoengineering of yeast and the expression of therapeutic glycoproteins with humanized N-glycosylation structures have shown significant promise (Wildt and Gerngross, 2005). Conveniently, yeast may be used as a powerful tool for structure–function analysis of membrane proteins, because many yeasts also contain an identical secretory pathway and many of the second messenger signaling pathway that exist in higher eukaryotic cell (Yin Et al., 2007).