Pichia pastoris 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 large positive eukaryoticcells, which is about 9.6 (\(\pm\)0.2) Mpb, organized into 4 chromosomes and around 5313 coding genes \cite{espejo2016}, however, this strain excells over other types of host, as it has a relatively fast growth rate in culture media that are composed of a carbon source, whether glucose, glycerol or methanol, the latter is the most frequently used \cite{calzada}, because it requires minimal, simple and economical means for the production of recombinant proteins and cell growth, such as effects of methanol concentration, effects of dissolved oxygen concentration, effects of induction temperature, effects of pH and effects of 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 such a copy number effect that the higher the number of expression cassettes, the greater the amount of protein product produced \cite{Vedvick_1991}.
2.3. Conditions necessary for the production of recombinant proteins 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, for this:
2.3.1. Effects of methanol concentration
The presence of methanol as an energy source is important for the culture 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), so much so that if there is shortage or excess of methanol in the culture it would impair the transcriptional efficiency of AOX and cause the accumulation of formaldehyde by contact of the dissolved oxygen (DO), and as a result the rate of expression of proteins would be affected, as a result, 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 very important, because if very high temperatures are used, folding can occur incorrectly, causing the degradation of the same and hence the stress of the strain, facts that would cause a metabolic overload in it, so the use of optimal temperature is very important, because 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.