1. Introduction
Nowadays into a balanced daily diet two needs arise and consist in include functional and nourishing foods \cite{Bhat_2011}. However, currently there is a significant population that suffers certain limitations when consuming some foods due to they generate an allergic reaction \cite{america2015}. Within the market are products where it is excluded those allergic ingredients and they are substituted by other ones that have some similarities such as vegan food which no contains milk either egg. Also, these two are mostly affect to children with 5 -8 years old, and they tend to be outgrown \cite{europe2013}. This trend was even coupled in other industries such as cosmetics and nutraceuticals which are named as hypoallergenic \cite{2011}. There are no clear proposals that aim to promote the consumption of these food into a final product, even without any improvement that has been achieved with biomolecular science modifications to reduce their allergic effect.
One of those daily foods that most of the people consume is egg. This is constituted by 55% of egg white \cite{deepak2018}, and contain certain proteins that cause allergy. The ovalbumin composes around of 65% of white, and is characterized by a sequence of amino acids that generate this allergic reaction \cite{huevo2009}. The database created by National Institute of Allergy and Infectious Diseases reports almost the total ovalbumin chain is allergic, defining 298 epitopes as possible source of allergy. In 2003, \cite{Mine_2003}, determined along the chain there are five epitopes as the several sites that promotes the allergy. When the body tries to digest the entire protein, it recognizes like a threat, thus the body activates the antibody agents as a red flag. This causes allergic symptoms such as a rash, redness on the skin, and even closure of the trachea, reducing the ability to breathe \cite{europe2013}.
Ovalbumin is a phosphoglycoprotein constituted by 368 amino acids and is generated in the oviduct of avian animals due to the located glandules in “magnum”, which secretes the constituents of the egg white. It is part of the serine proteins, but its main characteristic is not to inhibit proteases, as this type of protein generally does \cite{Huntington_2001}. Its real function in avian is not clear till this moment. However, it is consumed due to its high content part into the egg white and it was demonstrated that it can support some functions the serum albumin accomplishes in the human body. For instance, the transport of physiological and non-physiological substances that are not soluble in water, the maintenance of oncotic pressure, and the contribution of buffering capacity of the blood and even maintain albumin levels in people who have chronic kidney disease.
Based on the first report of the current project, which was determined the opportunity of the design of a product based on inclusion of a modified ovalbumin into a food daily product. This modification was developed with the exchange of some similar amino acids according with their hydrophobicity, and that are located on those five epitopes that were reported by \cite{Mine_2003}. The corresponding modifications showed that the tertiary conformation of the protein was maintained with respect to the original.
Also, the prediction of modified and original protein location showed a high probability of being extracellular as well as transmembrane location in 30-50 amino acid positions. The first indicates the molecule have hydrophilic radicals \cite{karp2011biologia}, and the second involves fulfilling functions such as being receptors that recognize and fix some molecules called ligands, channels or transporters of ions and solutes through the membrane and identifying nutrients, hormones or neurotransmitter \cite{karp2011biologia}. Finally, with this proposal the proteins structure was preserved which indicates the active sites are expected the same and it could be accomplished as the enhanced protein that will be added in a food product with a decrease in its allergic effects.
In this part of the project is proposed the microorganism and its production pathway to guarantee the fulfillment of having a modified ovalbumin produced and included into a daily product. Therefore, there was considered as Pichia Pastoris as the adequate microorganism to produce it based on recombinant technology. This consisted in determining the coding DNA sequence, the sequence expressed in codons, the RBS suitable for expressing GALNS, and making the cloning Fdesign.
2. Theoretical framework
The heterologous or recombinant protein production depends on the host to choose to produce the protein interest. This process consists in the isolation of the gene and subsequently cloned in an expression vector \cite{pazdernik2015}. There are different organisms which act as the host such as animal cells, yeast, bacteria or fungus. However, their performance will depend on the preference in which codons are formed for each amino acid \cite{pazdernik2015}. The protein expression system could be prokaryotic or eukaryotic. Prokaryotic is the renamed Escherichia coli, however this bacterium as prokaryotic cell microorganism has some limitations when a heterologous protein is going to be produced \cite{xu2014}. Eukaryotic cells facilitate the postranslational modification process, like the glycosylation, for the proteins that need this. Therefore, the cultures of yeast, insect cells, microalgae, filamentous fungus, and mammalian cells can handle it\cite{pazdernik2015,xu2014}.
Human proteins are characterized for their postranslational modifications such as glycosylation, phosphorylation, acylation, carboximetilation, and others \cite{1998}. Most of the therapeutic proteins that are in the market were obtained through a eukaryotic system that permit those protein modifications\cite{xu2014}. Mammalian cells is the first choice due to its ability to achieve adequate needed modifications, however, its maintenance is difficult due to it is very susceptible with environment changes and the treatment that they are exposed. In the last decade, yeast is the next option that which has been implemented to the recombinant human proteins, and the first selected fungus strain was the Saccharomyces cerevisiae in 1980, but some works affirm this has a unstable plasmid, low yield recorded, and hyperglycosilation phenomena \cite{calzada,sod}. In contrast, Pichia pastoris a methylotrophic yeast that has demonstrated its high yield production in the proper form for those therapeutic molecules. Due to its feeding source (methanol) most of the expression system are based on inducible promotes for methanol. Some of those recombinant proteins that have been cloned and expressed in this microorganism are: Kunitz inhibitor proteases, C fragment of tetanic toxin, human interleukin II, human lysozyme, human serum albumin, and among others \cite{calzada}.Its high quality of performance is supported by the respiratory metabolism allows to be achieved enormous cellular densities in the bioreactors \cite{Chen_2012}.
2.1. Pichia Pastoris
Pichia Pastoris is a single-celled eukaryotic organism called methylotophan yeast, which has a particular taxonomy, such that the genus Pichia is part of the family Saccharomycetaceae, the order Saccharomycetales, the Class Saccharomycetes, the Ascomycota phyleus and the Fungis kingdom, which is characterized by forming creamy, white and well-defined colonies. It is a highly used yeast as an expression system for the production of recombinant proteins, both for basic research and industrial purposes, this due to its easy genetic manipulation, its high levels of intra and extracellular production of the protein of interest and its ability to make post-translational modifications similar to those of higher eukaryote organisms; generating a correct folding of the protein \cite{guerrero-olazarn}.
This yeast has become one of the most important expression systems in the production of proteins, because it has a strong promoter and controlled which is the enzyme alcohol oxidase (pAOX1). It uses methanol as a source of carbon and energy and posses histidinol dehydrogenase gene (his4) for the synthesis of the amino acid histidine \cite{calzada}. Therefore, as a eukaryotic cell Pichia Pastoris yeast has many of the advantages of higher eukaryotic expression systems, such as protein processing, protein folding, and post-translational modification which makes it easier to manipulate. It is faster, easier and less expensive to use than other eukaryotic expression systems and has the additional advantage of generating expression levels of heterologous proteins between 10 and 100 times higher. These characteristics make Pichia very useful as a protein expression system \cite{invitrogen2010}.
2.2. Pichia Pastoris Characterization
Microscopically are positive large eukaryoticells, with a lenght of 9.6 (\(\pm\)0.2) Mpb, organized into 4 chromosomes and around 5313 coding genes \cite{espejo2016}. However, this strain highlights over other types of host due to its relatively fast growth rate in culture media. The culture is mainly composed of a carbon source, whether glucose, glycerol or methanol, and the last one is the most frequently used \cite{calzada}. Moreover, it requires minimal, simple and economical factors in recombinant protein and cell growth, such as effects of: methanol concentration, dissolved oxygen concentration, induction temperature, pH and nitrogen concentration \cite{guerrero-olazarn}.
On the other hand, expression systems require a method of transferring the DNA sequence of interest to the host cell together with a promoter capable of controlling the production of the foreign genetic product. Successful promoters have very high transcription efficiency, and these are very cheap \cite{Vedvick_1991}, however, most of the foreign protein genes expressed in P. Pastoris strains have shown high copy numbers and numbers and higher number of expression cassettes. Thus cassettes. Thus greater amount of protein are produced \cite{Vedvick_1991}.
2.3. Necessary conditions for the recombinant protein production using the P. Pastoris
To obtain high levels of modified ovalbumin in the crop, the strain is required to have the optimal conditions of substrate, dissolved oxygen, temperature, pH and nitrogen that allow the protein to be developed properly.
2.3.1. Effects of methanol concentration
The presence of methanol as an energy source is important for the culture medium medium because the transcription levels of the heterologous protein depend on the amount of methanol presented by the expression system of the enzyme alcohol oxidase (AOX). If there is shortage or excess of methanol in the culture it would impair the transcriptional efficiency of AOX and it cause the accumulation of formaldehyde by contact of the dissolved oxygen (DO). A possible result the protein expression rate would be affected. Mayson, Kilburn and their co-authors in 2003 suggested that the percentage rate of methanol in the culture medium for heterologous proteins varies from a range of 0.1 to 3.0 % (v/v) , since the methanol feeding system at growth-limiting rates could be 3 to 5 times higher than with excessive methanol feeding \cite{calzada,Mayson_2002}.
When starting the methanol feed, the gene is rapidly and fully transcribed. The strength of the promoter is demonstrated by the observation that the enzyme AOX comprises up to 30% of the soluble protein in extracts of P. pastoris grown in methanol \cite{Vedvick_1991}.
2.3.2. Effects of dissolved oxygen concentration
The use of methanol in the presence of oxygen is the first step in the assimilation of carbon sources, as well as in obtaining energy from it, generating with it the formation of formaldehydes [CH3OH-CH2O], a chemical that increases the production of recombinant proteins \cite{GAO_2013}, for this reason it is important to note that the metabolism of methanol in the presence of large quantities of P. Pastoris cultures results in an increase in the demand for DO, so that when cells grow under limiting conditions of DO decreases production levels of the heterologous protein \cite{calzada,Mayson_2002}; for this reason there are several strategies that can be used to maintain the ideal concentration of DO in the culture medium, such as \cite{calzada}:
- Increasesr airflow.
- Increasesr the rpm of the stirring process, which generates more oxygen in the culture medium.
- Cultivar la cepa P. Pastoris at temperatures below 30 °C.
- Increasers the ratio of the Air/O2 mixture.
- A use the ratio of the mixture of O2/N2.
- Controls methanol de feeding.
However, the dissolved oxygen ideonee concentration in the culture medium can range from 20% to 30% \cite{calzada,GAO_2013}.
2.3.3. Effets of indution temperature
The effect of temperature on cell growth and the production of recombinant proteins on the endoplasmic reticulum of yeast, can be relevant due to if very high temperatures are used, folding can occur incorrectly, causing the degradation of the same and hence the stress of the strain. This would cause a metabolic overload in it, so the use of optimal temperature is very important. The feeding of the substrate during the growth of the strain and methanol induction phases is 30°C, the metabolic stress of P. Pastoris and the formation of toxic products decrease significantly, causing cell growth and the production of recombinant proteins to increase \cite{calzada}. On the other hand, some research has reported that reducing the induction temperature of methanol from 30°C to 20°C is beneficial for the production of recombinant proteins, because at lower induction temperature, activation of alcohol oxidase (AOX) would increase the oxygen uptake rate (OUR), alleviating cell skeleton lysis and the secretion of protease, proteolytically reducing extracellular activity. However, doing this temperature drop and increasing OUR could be an inconvenience in the industry, as this increase would be a problem in terms of energy and as a result economic losses \cite{GAO_2013}.
Different studies have shown that the growth temperature of Pichia pastoris is 28 - 30°C for liquid cultures, plates, and slants. Growth above 32°C during induction can be detrimental to protein expression and can even lead to cell death \cite{invitrogen2010}.
2.3.4. Effects of pH
The P. Pastoris strain has a wide pH range to which they can be used, these can range from a pH range of 3.0 to 7.0, however, the optimal pH for strain and methanol growth as an inducer for the production of heterologous proteins is approximately 3.5 and 5.5, depending on the nature of the recombinant protein, for this, the pH has to be adjusted during the growth of the strain to a pH of 5.0 and this has to be adjusted again to a pH of 4.5 to introduce the methanol \cite{calzada,GAO_2013}.
2.3.5. Effects of nitrogen concentration
Nitrogen concentration is generally used to control the pH of the crop and for this, Yang and his co-authors in 2004 determined that the production of recombinant huridine by ammonium hydroxide (NH4OH) was obtained at a concentration of 0.4 M \cite{calzada,Yang_2004}.
3. Methodology
3.1 Determine the DNA sequence coding for the selected protein
The production of the recombinant protein is carried out through the eukaryotic organism Pichia Pastoris, due to the advantages it has such as the processing and folding of proteins, in addition to its easy manipulation for it. On the other hand, the system that uses Pichia Pastoris as a host is simpler and less expensive than others \cite{2000}. The protein of interest to be manufactured is Ovalbumin, the modified sequence of amino acids for Ovalbumin \cite{2020} is expressed below (see Figure \ref{175219}) and corresponds to the approach developed in the first delivery.