WO2008081291A2

WO2008081291A2 - Yeast, recombinant pig lactoferrin, expression vector of recombinant pig lactoferring gene, recombinant pig lactoferrin and production processes thereof.

Info

Publication number: WO2008081291A2
Application number: PCT/IB2007/004099
Authority: WO
Inventors: Cesare Pasquini
Original assignee: Cesare Pasquini
Priority date: 2006-12-28
Filing date: 2007-12-27
Publication date: 2008-07-10
Also published as: WO2008081291A3; ITBO20060891A1

Abstract

A production process of an expression vector, pPIC9-pLF,in which the pig lactoferrin gene is integrated, comprising following phases: - extracting the RNA of the gene of pig lactoferrin; - obtaining the cDNA of the gene of the pig lactoferrin from the RNA; and - integrating the cDNA of the gene of the pig lactoferrin in an expression vector pPIC9.

Description

YEAST, RECOMBINANT PIG LACTOFERRIN, EXPRESSION VECTOR OF RECOMBINANT PIG LACTOFERRING GENE, RECOMBINANT PIG LACTOFERRIN AND PRODUCTION PROCESSES THEREOF.

In agreement with European Community Rules, antibiotics must be progressively eliminated from feedstuffs destined for animals.

The present invention concerns a method for producing a protein which constitutes an effective and advantageous alternative to the use of antibiotics in animal food. This method further includes the use of a new genetically modified micro-organism which can express the protein and a new expression vector which enables genetic modification thereof. Further described are methods for obtaining the genetically modified organism and for obtaining expression vector of the invention. Further, the invention provides foods and/or food integrators for animals containing the protein and/or the micro-organism of the invention. DESCRIPTION OF THE PRIOR ART

It is known that molecules making up living organisms are often very complex organic compounds, and are in the majority polymers. For the aims of the present invention, proteins will be exclusively dealt with, which are polymers formed by chains of amino acids which perform many and varied functions in the organism. Proteins vary among themselves both in terms of the amino acids involved in the formation of the structure, and in terms of the monomeric units, and also exhibit very different properties in relation to the various three-dimensional structures they form.

The particularity of the proteins is that they carry out highly specific functions (for example enzymes, which precipitate specific chemical reactions by "selecting" the starting reactants and enabling the formation of a product having precise physical chemical properties), for which reason very complex molecular structures are necessary, in which structures both the amino acid sequence and the final three- dimensional conformation must be perfectly defined.

Normal protein synthesis internally of the cells is performed by means of very complex mechanisms, fruit of natural evolution, in which the DNA, the genetic information bearer, regulates the formation of the proteins with specific functions, ensuring correct amino acid composition and the right three-dimensional arrangement of the molecule. From the above, it can easily be understood how difficult artificial synthesis of the complex molecules of this type is, given that not only is it necessary_Nto respect the programmed amino acid sequence, but also to guarantee correct arrangement of the formed macromolecule.

This requires many intermediate passages and many purification phases inevitably leading to a considerable increase in production costs. Exploitation of the production of these molecules by living organisms enables products to be obtained with a single passage, and starting from very simple initial reactants.

It is known that single-cell organisms (bacteria and fungi), notwithstanding their small size, are very special chemical "reactors", being able to synthesis complex molecules starting from simple compounds which can be found in the environment they live in.

The growth and proliferation of the micro-organisms, often known as "biomasses" in fermentation processes, is a factor of special importance when their metabolic activity is to be exploited in order to produce substances of interest: the quantity of substance produced obviously depends on the number of micro-organisms which produce it.

As in all eco-systems, in order to enable micro-organisms to survive, the environment must offer ideal conditions, so that it is not sufficient to make nutrients available - other factors are also required (such as pH, availability of oxygen for respiration and absence of toxic substances) which must be kept constant over time. It is therefore possible to exploit the metabolic activity of the micro-organisms with the aim of producing substances of interest, limitedly however to those compounds which the organisms normally produce in nature in order to satisfy their needs. New DNA cloning and heterological complex molecule expression technologies have helped to overcome this drawback, as they identify the fragment of cDNA (or gene) which is responsible for the production of the complex molecule present in superior organisms. The insertion thereof into micro-organisms is done by means of recombinant expression vectors. The micro-organisms thus modified can produce complex molecules, such as proteins. Yeasts are the type of micro-organisms mostly used for this purpose. It is known that by the term fermentation any process is intended in which the micro-organisms rapidly multiply, causing the transformation of the mixture of reaction. The metabolic activity of the cells causes enrichment of the mixture of complex organic molecules (among which the substance which is to be produced) starting from simple constituents (nutrients). In order for this process to take place the micro-organisms must be provided with an environment in which they can prosper: thus, nutrients must be available, the pH and the temperature must be suitable and a correct gas exchange must be maintained with the outside in order to ensure respiration. Usually the fermentation process can be schematised as follows:

For industrial production, usually discontinuous or batch processes are used, as in the above diagram.

The salient phases are represented by creation of the correct environment for the fermentation, by the insertion of the specially-selected micro-organisms and by the final recuperation of the formed products. The environment and the operating conditions have to be such as to enable rapid development of the bacterial colonies, such that the subsequent protein production is effective.

In order to have a homogeneous means of growth all the mixture must be continuously shaken. In order to ensure good gas diffusion and a sufficient concentration of nutrients at each point, production of surface foam must be prevented, as this would prevent gas exchange. For this purpose, often anti-foam agents are used which enable a continuous gas exchange without interfering with the fermentation process.

In addition to the main nutrients, many other substances can be inserted into the process, in small concentrations, which substances are indispensable for the cellular metabolism; in particular, diluted salt solutions can be used.

The insertion of the micro-organisms which are responsible for the fermentation, known as inoculation, is performed by placing some selected colonies in the fermentation medium, which colonies are grown in a special medium: the colonies represent the initial biomass. On completion of the biomass growth phase, in which the micro-organisms use the nutrients present in the environment in order to grow in size and multiply, the production of the complex organic compounds (such as for example proteins) begins, which complex organic compounds accumulate in the fermentation medium. This phase can be solicited from the outside in order to produce the substance of interest, for example through variations in the nutrients or by the introduction of substances which activate the specific promoter controlling the gene of the protein production.

In the fermentation mixture, therefore, the following will be present: the microorganisms, the substance of interest and all the other metabolites produced, as well as the non-utilised nutrients. The products of interest must therefore be extracted from the mixtures.

In consideration of the above, the present invention is inserted in the technical sector concerning the production of lactoferrin, which is especially useful for integrating animals' (for example piglets') diets during weaning or precocious weaning. It is known that lactoferrin (LF) is a basic glycoprotein, belonging to the transferrin group, a class of non-heme proteins which linkiron at pH-neutral and pH-alkaline and release iron at a lower pH.

- Lactoferrin is synthesised in the mammary gland and in other exocrine glands and is stored in the granules of the polymorphonucleate leucocytes;

- it is involved in the metabolism of iron;

- it modulates the inflammatory response;

- it regulates cellular growth andcellular differentiation;

- it is a bioactive milk peptide. At birth piglets are immature from the digestive and immune point of view; on weaning they suffer from numerous stressful factors (milk-feeding by the mother, formation of new groups, health treatments, etc.).

It would therefore seem of considerable interest to integrate a premature piglet's diet with bioactive molecules having nutraceutic values and being species- specific, which can support the immune deficiencies of the animal.

Considering the notable difficulty of colonisation, in the first hours of life, on the part of the useful bacterial flora, the lactoferrin takes on an indispensable importance in maintaining a correct balance of the intestinal bacterial flora. The interest in this protein in the field of pig rearing is due to its nutraceutic action; a nutraceutic is a bioactive substance which does not have any nutritional value, but which contributes to improving the performance of the organism. Lactoferrin has an inhibiting action on pathogens and therefore the efficiency of the immune system; it is known that during the phase of weaning the piglet is particularly subject to onset of pathologies which derive from immunitary deficits caused by stress and by lowering of the defences passively acquired from the mother in the colostrum and milk.

Further, lactoferrin promotes assumption of iron at enteric level, as it interacts specifically with a receptor located on the brush border of the intestinal epithelium.

The production of pig lactoferrin by artificial synthesis is extremely complex. The possibility of obtaining recombinant lactoferrin via heterologous expression is therefore extremely interesting.

The sequence of cDNA which is responsible for the production of pig lactoferrin was identified by Alexander, L. J., et al., Animal Genetics, 23:251-256 (1992): the sequence is2259 nucleotides long, codes for a leader peptide 19 amino acids long and for a mature protein of 684 residual amino acids weighing 78 KDa. The pig lactoferrin gene exhibits 257 bases in the 3' non-translated region, the polyadenylation signal in nucleotide position 2273-2278 and the polyadenylation tract (A15) at nucleotide 2308 position (Wang et al., 1997).

Also known is that Pichia pastoris is a methylotrophic yeast which can exploit methanol as a single source of carbon and energy, and which is used as an expression system for production of heterological proteins.

Yeasts as an expression system have the following basic advantages:

- high safety level;

- rapid growth; - easy manipulation of the genetic patrimony. SUMMARY OF THE INVENTION

The aim of the present invention is to provide a fermentation process for Pichia pastoris with which pig lactoferrin production can be induced and obtained via heterological expression. A further aim of the invention consists in actuating the above-cited fermentation process using simple and inexpensive organic compounds. A further aim of the present invention is to provide a new expression vector which includes a pig lactoferrin gene and a process for producing it. In a further aspect of the present invention the applicant provides a new recombinant Pichia pastoris strain which can advantageously express pig lactoferrin and a process for producing the recombinant strain. Also objects of the present invention are recombinant pig lactoferrin obtainable with the process of the invention, feeds or feedstuff integrators for animals, in particular for piglets, containing recombinant pig lactoferrin and/or the new strain of Pichia pastoris obtainable with the production methods of the present invention. The above-cited aims are obtained in accordance with the content of the claims. The characteristics of the invention are described in the following, with special reference to the considered examples. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a map of vector pPIC9

Figure 2 is the multiple cloning site of pPIC9

Figure 3 is a diagram of the gene substitution at AOX1 in Pichia pastoris GS115.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The production process of the expression vector of the invention in which the pig lactoferrin gene is integrated into base vector pPIC9 comprises the following phases:

- extracting the RNA of the pig lactoferrin gene;

- obtaining the cDNA of thepig lactoferrin gene from the RNA; and

- integrating the cDNA of the pig lactoferrin gene into the pPIC9 expression vector.

A second expression vector is obtained, which the applicant has termed pPIC9- pLF, which is constituted by the base vector pPIC9 in which the pig lactoferrin gene is integrated. The production process of the recombinant Pichia pastoris strain which can express pig lactoferrin comprises the following phase:

- integrating the expression vector pPIC9-pLF into Pichia pastoris GS115, obtaining a new strain of Pichia pastoris, which the applicant has called Pichia pastoris RLF9. Pichia pastoris RLF9 is therefore constituted by Pichia pastoris SG115 in which the expression vector pPIC9-pLF has been integrated.

The production process of pig lactoferrin according to the invention comprises the following phase:

- inducing the pig lactoferrin expression by adding methanol to the Pichia pastoris RLF9 culture.

The pig lactoferrin thus produced was analysed using SDS-PAGE and Western blotting techniques: in the latter technique the pig lactoferrin was recognised by an rabbit anti human primary antibody directed against human lactoferrin. Research carried out by Magnuson et al (1990) has demonstrated that human and pig lactoferrin share antigenic determinants and present a considerable homology of the tertiary structure.

L¹SDS-PAGE and the Western blotting method evidenced the presence of two bands of different molecular weight (about 80 and 50 Kda). Pig lactoferrin has two N-glycosylation sites and three potential O-glycosylation sites; the deglycosylated protein weights about 77 KDa. The larger band would therefore represent the glycosylated form of the pig lactoferrin, as the glycosylated residues can significantly increase the molecular weight of a protein. Glycosylation constitutes a process of fundamental importance, as the biological effectiveness of the lactoferrin in general is linked to the presence of the glycosidic chains. It is known that the resistance to protease treatment and to the variations of pH is influenced by the level of glycosylation. The band having lower molecular weight, on the other hand, probably represents a part of the protease-processed lactoferrin. The extraction of the RNA from the pig lactoferrin can be done by using sample tissues from pig exocrine glands, advantageously sow mammary glands, using commercially-available cell lysis reactants. The sample is preferably homogenised and the extraction is preferably performed using TRIZOL® (cell lysis solution marketed by Invitrogen).

The cDNA was obtained by reverse transcription of the corresponding RNA and the gene of interest was amplified using the cDNA PCR technique. The applicant obtained pPIC9-pLF, which was used as the expression vector for directing secretion of proteins in the culture medium. To obtain the expression vector the applicant integrated the pig lactoferrin gene into the pPIC9 plasmid, commercially available from Invitrogen. This plasmid, the map of which is reported in figure 1 , possesses the codifying sequence for the N-terminal (a-factor) secretion signal upstream of the multiple cloning site. The vector possesses the alcohol oxydase AOX1 promotor, which enables expression of high levels of protein by induction of the methanol, and the gene His4, which enables growth in histidine-free medium. The expression vector linearised pPIC9-pLF was integrated into the genome of the host and promotes stable maintenance of the gene of interest. Further, the vector contains the gene for resistance to ampicillin for selection in E.coli.

It is advantageous to multiply the expression vector pPIC9-pLF by integrating it in a biological system which can multiply, such as an Escherichia coli bacteria, before integrating it in the pre-selected Pichia pastoris strain. For this purpose advantageously use can be made of strains of Escherichia coli of the following types: XL1-Blue, DH5α and JM 101 , preferably DH5α and JM 101. The integration of the construct in the bacteria can be done by electroporation or by transformation using the "HEAT SHOCK" method. After growing the Escherichia coli recombinant strain in which the pPIC9-pLF construct has been integrated, and after selecting on culture media containing ampicillin, the plasmid DNA was extracted from the selected cultures. The extraction of the plasmid DNA can be done using various techniques; by alkaline lysis, boiling lysis, or using special commercially-available kits. The linearised DNA was then integrated in the GS 115 Pichia pastoris cells by electroporation. The Pichia pastoris strain used is a methyltrophic yeast which, in the absence of a source of repressive carbon, such as glucose, is able to use methanol as a source of carbon. The promotor of the alcohol oxydase (AOX1) controls the expression of this enzyme, which catalyses the first stepin the metabolic pathway of the methanol. In the absence of methanol no alcohol oxydase can be detected. Further, the strain of Pichia pastoris GS115 is the carrier of a mutation in the gene of the histidinol dehydrogenase (his4), which makes it unable to synthesise histidine. All the plasmids which bear the HIS4 gene complement the mutation of the host and the transformers are selected for their ability to grow in a histidine- free medium. The linear plasmid DNA generates stable transformers of Pichia pastoris through homologous recombination with the homology regions of the genome. Linearisation of the DNA with Sad promotes the event of genie insertion of the locus AOX1 ; this determines the phenotype His+Mut+ in the his4 GS115 strain. (FIG. 3)

The recombinant strain of Pichia pastoris RLF9 obtained, in which the pig lactoferrin gene has been inserted, is thus selected from an inoculation on MGY

(minimal glycerol medium, without histidine) before proceeding with the fermentation and the consequent heterological production of pig lactoferrin.

CREATION OF THE CORRECT FERMENTATION MEDIUMOF PICHIA

PASTORIS RLF9

It is advantageous to perform fermentation of Pichia pastoris RLF9 using an mediumin which glycerol is present as a main nutrient, together with the substances which render the means suitable for growing the colonies of Pichia pastoris RLF9.

The culture medium, termed FBS (fermentation basal salts) has the following composition.

The culture medium is sterilised at 121⁰C for 20 minutes; is preferably brought to pH 5.0 with a 25% ammonia solution (NH₃); a saline solution (PTM) is added, providing other substances indispensable for metabolism of the Pichia pastoris, in quantities of 4.35 ml/litre of FBS medium. The PTM saline solution, also sterile, has the following composition.

The growth medium, as above-prepared, is inserted into the reactor (fermenter) and the conditions defined as fermentation parameters are advantageously set: temperature 30⁰C - oxygen >20% pH 5.0-6.0 relative to first and second phase shaken at 500-1500 rpm (rpm) aeration 0.1-1.0 litres of oxygen/litre of growth medium/minute. The cells thus can grow at the ideal temperature, while the availability of nutrients and the gas exchange are guaranteed by shaking the medium. This condition might lead to the formation of foam, with drawbacks that are known to the expert in the field; for this reason, polypropylene glycol (PPG) is used as an anti-foaming agent, for example.

Once the fermentation medium has been prepared, the Pichia pastoris RLF9 is inserted (inoculated).

The pH is advantageously continuously controlled during the progress of the fermentation and kept to a constant value by introducing phosphoric acid and ammonia, or phosphoric acid and potassium hydroxide. INSERTION (INOCULATION) OF PICHIA PASTORIS

The cells of Pichia pastoris RLF9 which are suitable for heterological production of lactoferrin are priorly developed on a special culture medium and when they have reached the desired growth level are inserted in the fermentation medium in which they will continue to develop and reproduce up until they reach the ideal mass for enabling lactoferrin production.

-To develop the desired inoculation (of Pichia pastoris) an MGY medium is used (inoculated with a colony from an MGY plate or from a glycerol stock). - The MGY inoculation medium is composed as follows. MGY Minimal Gl cerol medium without histidine

PROCESS OF FERMENTATION

This begins with a biomass growth phase, known as the batch with glycerol phase, lasting about 18-24 hours.

During this phase the cells introduced, preferably at a quantity of 5-10% of the total volume of the reactor, with a discontinuous process, consume the nutrient (glycerol) in the fermentationmedium in order to and multiply. By monitoring the gases which are produced in thefermentation medium it can be established when this phase is completed (the oxygen value increases by 100%).

The process proceeds with a second feeding phase with a 50% glycerol solution, added to at a 12 ml/litre rate with PTM, at a dose of 18 ml/hour/litre of the initial volume for a duration of at least 4 hours.

During this phase the glycerol introduced into the reactor is consumed; the consumption is evidenced by an increase in the value of the oxygen of about

100%.

At the end of the second phase, the biomass reaches about 180-220 g/l, control of the above-mentioned value can be performed by removal of a 10 ml sample.

During this second phase, the value of the oxygen can lower to below 20%; in this case the feeding is stopped (introduction of nutrients), the oxygen value is replenished without varying the other parameters, and feeding is recommenced, possibly varying the shaking parameters or the volume of air introduced into the mixture.

In the third and last phase production of lactoferrin is induced by changing the nutrient, more precisely by using methanol.

During this phase the source of carbon "methanol" activates the specific promoter which controls the gene for the production of pig lactoferrin introduced into Pichia pastoris.

The applicant has found that in the third phase the methanol must be introduced slowly into the reaction environment: a rapid addition would lead to the formation of zones having too-high a concentration of nutrients, which would result in killing the cells.

Preferably the third phase, feeding with 100% methanol, added to at a rate of 12 ml/litre of PTM, is divided in turn into three sub-phases, listed in the following table.

During sub-phase A the methanol tends to accumulate in the first 2-3 hours and the oxygen values measured are consequently variable; if the values fall below 20% the feeding is suspended and recommenced only when the oxygen has increased. The biomass must be adapted to the methanol for a duration of 2-4 hours (the time indicated in the table). During the third phase, overall, the quantity of nutrient (methanol) consumed can be expressed as 740 ml methanol per initial litre , and the biomass grows up until it reaches a value of about 350-450 g/litre.

During the third stage the methanol, apart from functioning as a nutrient, and as previously mentioned, activates the specific promoter which controls the gene for the production of pig lactoferrin introduced in Pichia pastoris RLF9. The fermentation medium is enriched during this phase by the protein to be obtained.

It is advantageous to inhibit the protease activity, with the aim of not reducing the quantity of protein produced, slowly bringing the pH of the culture broth to about 3.0 at the end of the second phase or at the start of the third phase. This reduction in pH must be slow (4-5 hours) so as not to damage the metabolic activity of the cells.

The duration of the third phase is about 70 hours, during which 74% of the methanol introduced is consumed. The process of fermentation can at this point be considered complete, and one can proceed to the extraction of the recombinant pig lactoferrin produced in the fermentation medium using conventional techniques, for example ultrafiltration.

Alternatively Pichia pastoris RLF9 cells can be added to feeds and/or animal integrators. The advantages of the invention are obvious: starting from simple and inexpensive organic compounds (glycerol and methanol) a protein product can be obtained, namely pig lactoferrin, a compound the artificial synthesis of which would be extremely complex.

EXAMPLES Where not expressly indicated, the protocols followed have been taken from Current protocols in molecular biology (Ausbel et al, 1994), Molecular cloning, A laboratory manual (Sambrook et al, 1989) and a Pichia expression kit (marketed by Invitrogen).

The compositions of the solutions or culture media indicated in the processes are reported following the procedure.

TOTAL EXTRACTION OF RNA FROM THE SOW MAMMARY GLAND

• the sow mammary tissue (from 50 to 100 mg) is placed in a 1.5 ml tube and 1 ml of TRIZOL^® is added

• the sample is homogenised with a homogeniser, such that the solution is mixed with the tissue

• after a 5-minute incubation at room temperature to allow for complete dissociation of the nucleoprotein compounds, 0.2 ml of chloroform-isoamyl (24:1 ) are added, the tube is upturned several times for 15 seconds and incubated at room temperature for 2-3 minutes » the sample is centrifuged at 12000 rpm for 15 minutes at 4°C

• the supernatant is removed and transferred to a sterile tube

• 0.5 ml of cold isopropanol are added to the aqueousphase

• the sample is incubated for 10 minutes at RT

• the sample is centrifuged at 12000 rpm for 10 minutes at 4°C • the supernatant is removed

• 1 ml of EtOH 70% is added to the pellet

• the sample is centrifuged at 7500 rpm for 5 minutes at 4°C

• the supernatant is eliminated

• the sample is centrifuged for a few seconds and the EtOH is completely removed from the walls of the tube • the tube is left with the cap open for 5 minutes, until the pellet is dry

• the pellet is resuspended in 30 μl of DEPC (diethylpyrocarbonate) water

• part of the RNA extracted is placed at -80°C, while a portion (4 μl) is viewed on a formaldehyde gel. Before adding the sample buffer (10 μl) the RNA is incubated for 5 minutes at 65⁰C; after adding the sample buffer the

RNA is newly incubated at 65°C for 10 minutes. Thereafter, loading in the gel wells is performed.

One hour at a voltage of 130 V and a 200 mA current is allowed, viewing with ethidium bromide in UV light. The formaldehyde gel was prepared as follows: 1. 1 g of agarose was dissolved in 60 ml of water

2. under the fume hood, 12 ml of 40% formaldehyde was added

3. under the fume hood, 8.3 ml of 10% MOPS was added

4. the solution was poured into the special gel tray. Composition of the 10X MOPS • 0.2 M MOPS (3-[N-morpholino]propanesulfonic acid)

• 0.08 M sodium acetate pH 7

• 0.01 M EDTA

SAMPLE BUFFER (conserve at 4°C or -20⁰C according to the frequency it is used at)

• 5X MOPS 0.2 ml

• formamide 1 ml

• 40 % formaldehyde 0.35 ml

• ethidium bromide 10 μl

• loading buffer 200 μl LOADING BUFFER

• 50 % glycerol

• 1 mM EDTA pH 8

• 0.25 % bromophenol blue

• 0,25 % xylene cyanol FF

REVERSE TRANSCRIPTION OF RNA IN CORRESPONDING cDNA The reaction mixture is schematised in the following table:

^*diluted with H₂O DEPC

^**this amount derives from the previous precipitation and resuspension in 5 μl of

H₂O DEPC of the total extracted RNA.

The reverse transcription cycle used is composed of the following phases:

• a 5-minute stage at a temperature of 70°C for; in the first phase only the buffer, the primer and the total RNA are added • a stage at a temperature of 42°C for 65 minutes; the enzyme and the dNTPs (deoxyribonucleotides) are added

• a stage at a temperature of 70⁰C for 15 minutes

• a stage at a temperature of 4°C for an unspecified time

To amplify the cDNA of the gene, the following PCR reaction was set up, with the following composition:

The amplification conditions used are the following:

_ a phase at a temperature of 94° C for 5 minutes _ 40 cycles composed of the following phases: . • a phase at a temperature of 94°C for 30 seconds

• a phase at a temperature of 60⁰C for 1 minute

• a phase at a temperature of 72°C for 3 minutes and 30 seconds _ a phase at a temperature of 72°C for 7 minutes _ a phase at a temperature of 4°C for an unspecified time MANIPULATION OF THE DNA DIGESTION WITH RESTRICTION ENZYMES Digestion is performed as follows:

The mixtures were left at 37°C for about 2 hours or overnight, according to the enzyme cut effectiveness.

SPLICING

T4 DNA ligase unites the restriction fragments of DNA which the blunt and cohesive ends exhibit, catalysing the formation of phosphodiester bridges between the 5' phosphate group and the 3' hydroxyl terminal in the two-filament DNA. The reaction mixture is made as follows:

AGAROSE GEL ELECTROPHORESIS

The agarose gel is used to separate the DNA fragments according to size and degree of super-winding. The agarose concentration used is 0.8%.

Ethidium bromide is added to the gel, in a concentration of 1 μl/50 ml of gel (stock mg/ml); this is a DNA-intercalation compound and is used to enable the DNA to be viewed when the gel is illuminated with UV rays in the transilluminator.

Before loading the DNA is mixed with a loading buffer which increases the density of the DNA solution and enables its lowering into the gel wells.

The voltage is set at 85-90 V.

The buffer used is TBE 0.5X, prepared starting from TBE 10X.

TBE 10X (1000 ml)

• TRIS 108 g

• Boric acid 55 g

• 0.5 M EDTA pH 8 4 ml

LOADING BUFFER

• 0.07 % bromophenol blue

• 50 % glycerol

GENE CLEANED OF FRAGMENTS EXCISED FROM THE GEL

To elute the DNA fragment fromthe gel, extraction kit QIAquick marketed by Quiagen was used, operating in the following way:

1. the bands are excised with a clean forceps and placed in a 1.5 ml tube and weighed.

2. three volumes of Buffer QG are added for each gel volume, considering that 100 mg of material correspond to about 100 μl.

3. leave for 10 minutes in incubation at 50⁰C, such that the gel completely dissolves. To facilitate the dissolving of the gel, the tube is vortexed each 2-3 minutes during incubation. At the end of the phase the gel must be completely dissolved and it is important that the pH be less than or equal to 7.5, in order to facilitate the DNA bond to the resin.

4. the contents of the tube are transferred to the column supplied with the kit and are centrifuged for 1 minute at maximum velocity.

5. the elutedmaterial is removed the operation repeated.

6. the column is then washed with 0.75 ml Buffer PE by centrifuging for 1 minute at maximum velocity

7. the column is located in a clean 1.5 ml tube

8. the DNA is eluted by adding 50 μl of Buffer EB (10 mM TRIS-CI pH 8.5) and by centrifuging at maximum velocity for 1'

9. the DNA is then resuspended in sterile H₂O MQ.

DNA PRECIPITATION

The ligasemixture is precipitated not only in order to concentrate the DNA, but also to free it of the salts contained in the buffer which might interfere with the following transformation. • equal volumes of cold isopropanol and 1/10 volume of 3 M sodium acetate pH 5.5 are addedto ligase mixture

• the mixture is vortexed

• the mixture is incubated for about 30 minutes at room temperature (greater efficiency is obtained by incubating for 30 minutes or overnight at -20⁰C) • the mixture is centrifuged at maximum velocity for 30 minutes (preferably in a refrigerated centrifuge)

• the surnatant is carefully eliminated

• 50 μl of cold EtOH 70% are added in order to cover the pellet

• the mixture is vortexed • the mixture is centrifuged at maximum velocity for 30 minutes

• the surnatant is removed

• centrifuging is repeated for a few seconds more in order completely to eliminate the alcohol from the tube walls

• the pellet is dried at the Bunsen burner or in air for between 5 and 10 minutes

• on the basis of its consistency, the pellet is resuspended in sterile MQ TRANSFORMATION OF ESCHERICHIA COLI E.coli DH5α WITH THE HEAT SHOCK METHOD

A) PREPARATION OF THE COMPETENT CELLS ■ E.coli DH5α is grown overnight at 37°C in a shaker (250 rpm), i.e. a single bacterial colony of E.coli in 10 ml of liquid LB

^■ 5 ml of culture are transferred into a flask with 500 ml of liquid LB; this is incubated at 37°C in a rotary shaker (250 rpm) until OD₆oo =0.4 (about 3-4 hours) ■ the culture is transferred into 250 ml tubes and left in ice for 20 minutes ^■ the mixture is centrifuged at 4°C for 10 minutes at 3500 rpm

^■ the cells are re-suspended in 30 ml of 0.1 M cold CaCb and incubated in ice for 10 minutes

^■ the mixture is centrifuged at 4⁰C for 10 minutes at 3500 rpm ■ the surnatant is removed and the cells are re-suspended in 0.1 M CaCI₂ and 15% cold glycerol

^■ amounts of 140 μl are prepared; they are passed into liquid nitrogen and conserved at -8O⁰C.

B) HEAT SHOCK " the competent cells taken from the refrigerator at - 80⁰C are left in ice for

10 minutes

^■ 2 μl of DNA (about 50 ng/μl) are directly added to the cells • They are left in ice for 45 minutes

The mixture is incubated for 2 minutes at 42°C " the mixture is left in ice for 90 seconds

^■ 900 μl of LB liquid are added

^■ the mixture is incubated for one hour at 37°C

^■ 50 μl of the extender is combined with the remaining concentrate after centrifugation for 5 minutes at 12000 rpm ■ leave to grow overnight at 37°C in the thermostat

Composition of the LB (Luria-Bertani) Medium

Yeast extract 5 g

Triptone 10 g

NaCI 10 g H₂O bi-distillate 1 litre The medium is brought to pH 7.0 and sterilised in the autoclave at 115°C for 15 minutes before inoculation.

The growth and selection of the E. coli in which the recombinant vector pPIC9-pLF has been integrated is performed in LB with additional 50 mg/ml of ampicillin. EXTRACTION OF PLASMID DNA

The procedure followed for extraction of DNA from E.coli DH5α is illustrated in the following points; the composition of the solutions used are reported in the following:

1 ) collection of the bacterial cells: the E.coli cells are pelleted by centrifuging at maximum velocity for 5 minutes; the medium is delicately removed

2) resuspension of the cells: 4 ml of E1 solution are added to the pellets; the cells are re-suspended by vortexing, until the suspension is homogeneous

3) cellular lysis: 4 ml of E2 solution are added, followed by delicate but energetic mixing, until the lysate appears uniform 4) treatment with RNases: 10 μl of RNases (stock 10 mg/ml) are added and incubated for 20 minutes at 55^GC 5) neutralisation: 4 ml of E3 solution are added and the tubes are upturned 5 times to mix; vortexing must be avoided. The mixture is centrifuged at 2O⁰C at 15000 x g for 10 minutes 6) balancing the columns: the balancing of the anion-exchange resin column,

JETSTAR marketed by GENOMED, is done by percolating 10 ml of E4 solution in each column 7) loading the columns: the surnatant resulting from the preceding centrifugation is applied to the column and left to percolate by gravity 8) washing the columns: the columns are washed twice with 10 ml of E5 solution

9) elution of the plasmids: the DNA is eluted with 5 ml of E6 solution

10) precipitation of the plasmids: the DNA is precipitated with 0.7 volumes of isoprøpanol (3.5 ml). It is centrifuged at 4°C at 15000 x g for 30 minutes. The DNA is washed with EtOH 70% and centrifuged again. The pellet is left to dry in air for 10 minutes; the DNA is re-suspended in 50 μl of sterile H₂O. COMPOSITION OF THE E1 SOLUTION (cell resuspension)

• 50 mM Tris

• 1O mM EDTA-HCI pH 8.0 COMPOSITION OF THE E2 SOLUTION (cell lysis)

• 200 mM NaOH

• 1.0 % SDS (sodium dodecyl sulphate) COMPOSITION OF THE E3 SOLUTION (neutralisation)

• 3.1 M potassium acetate-acetic acid pH 5.5 COMPOSITION OF THE E4 SOLUTION (balancing the column)

• 60OmM NaCI

• 100 mM sodium acetate

• 0.15% TritonX-100-acetic acid pH 5.0 COMPOSITION OF E5 SOLUTION (washing the column) • 800 mM NaCI

• 100 mM sodium acetate-acetic acid pH 5.0 COMPOSITION OF E6 SOLUTION (DNA elution)

• 125O mM NaCI

• 10O mM ThS-HCI pH 8.5 TRANSFORMATION OF Pichia pastoris GS115 into Pichia pastoris RLF9 (a) PREPARATION OF THE COMPETENT CELLS

1. Pichia pastoris GS115 is grown in 5 ml of YPD in a 50 ml flask overnight in a shaker (250 rpm) (preinoculate)

2. 500 ml of fresh YPD are inoculated in a two-litre flask with 0.5 ml of preinoculate and left to grow overnight up to an OD₆oo of 1.3-1.5

3. The cells are centrifuged at 1500 x g for 5 minutes at 4°C

4. The pellet is re-suspended in 500 ml of sterile water cooled in ice

5. The cells are centrifuged as in point 3 and the pellet is re-suspended in 250 ml of sterile water cooled in ice 6. The cells are centrifuged as in point 3 and the pellet is re-suspended in

200 ml of 1 M sorbitol cooled in ice

7. The cells are centrifuged as in point 3 and the pellet is re-suspended in 1 ml of 1 M sorbitol for a final volume of about 1.5 ml

8. The cells are portioned (80 μl) and conserved at -80⁰C (b) ELECTROPORATION

1. (80 μl) 5-20 μg of linearised DNA are mixed with the cells in a maximum volume of 10 μl of TE.

2. The cells and DNA are transferred into a previously-ice-cooled 0.2 cm electroporator cuvette 3. This is left in ice for 5 minutes

4. The cells are electroporated and 1 ml of 1 M cold sorbitol is immediately added

5. The contents are transferred to the cuvette in a sterile tube and the cells are placed on MD medium in portions of 400 μl Composition of YPD (Yeast Extract Peptone Dextrose) Yeast extract 1O g

Peptone 2O g

Dextrose 20% 100 ml

H₂O bi-distillate 900 ml The dextrose 20% is added after having autoclaved the medium.

MD composition (Minimal Dextrose medium, without histidine)

YNB (Yeast Nitrogen Base) 13,4% 100 ml

Biotin 0,02% 2 ml

Dextrose 20% 100 ml H₂O bi-distillate 800 ml

The components of the sterile medium are added after having cooled the autoclaved H2O bi-distillate to 6O⁰C.

All of the above has been described by way of non-limiting example; any variations of a practical-applicational nature of the described phases defining the process of the invention are intended as falling within the protective field of the invention as above-described in as claimed herein below.

Claims

1 ) Yeast, recombinant pig lactoferrin, Pichia pastoris RLF9, constituted by Pichia pastoris SG115 in which expression vector pPIC9-pLF has been integrated.

2) Expression vector of pig lactoferrin gene, pPIC9-pLF, constituted by base vector pPIC9 in which the pig lactoferrin gene is integrated.

3) Production process of RLF9 strain of Pichia pastoris RLF9, comprising a phase of:

- integrating the expression vector pPIC9-pLF in Pichia pastoris GS115.

4) A production process of an expression vector, pPIC9-pLF, in which the pig lactoferrin gene is integrated, comprising following phases:

- extracting the RNA of the gene of pig lactoferrin;

- obtaining the cDNA of the gene of the pig lactoferrin from the RNA; and

- integrating the cDNA of the gene of the pig lactoferrin in an expression vector pPIC9. 5) A production process of recombinant pig lactoferrin comprising the following phase:

- inducing expression of pig lactoferrin by adding methanol to the Pichia pastoris RLF9 culture.

6) The process of claim 5) comprising phases of: - creating a sterile culture medium at a predetermined pH,

- inoculating cells of Pichia pastoris RLF9 in a fermenter containing the culture medium;

- actuating a first phase for growth of a biomass following consumption of glycerol by the cells of Pichia pastoris RLF9; - actuating a second phase for further growth of the biomass in consequence of the consumption of glycerol by the cells of Pichia pastoris RLF9; activating, in a third phase, and by means of methanol, further growth of the biomass and a specific promotor which control the gene for the production of pig lactoferrin introduced in the cells of Pichia pastoris RLF9; - extracting the pig lactoferrin from the fermentation medium.

7) The process of claim 6, wherein in the third phase methanol at 100% is fed, added to by a portion of saline solution, in a first sub-phase A), at a dose of about 3.6 ml/hour/litre initial volume, for a duration of between two and four hours, in a second sub-phase B) at about 7.3 ml/hour/litre initial volume for a duration of at least two hours, and in a third sub-phase C) at about 11 ml/hour/litre initial volume for a duration such as to identify an overall duration of the third phase of at least seventy hours.

8) The process of claims from 6 to 7 in which at an end of the second phase or at a start of the third phase the pH of the fermentation mediumis brought to a pH of about 3.0.

9) The process of claims from 6 to 8, wherein the inoculation of cells of Pichia pastoris RLF9 in the fermenter is comprised between 5 and 10% of the initial volume of the fermenter, in which the first phase includes glycerol supply at 4% with a duration comprised between 18 and 24 hours and in which the end of the first phase, following complete consumption of the glycerol, is identified by an increase in oxygen of about 100%.

10) The process of claims from 6 to 9, wherein the second phase includes feed of glycerol at 50%, added to by a portion of saline solution, for at least four hours, and in which the end of the second phase, consequent to complete consumption of the glycerol, is identified by an increase in oxygen by an amount close to 100%. 11 ) The process of claims from 6 to 10, wherein the culture medium is subjected to following fermentation parameters:

- temperature 30°c;

- oxygen > 20%; - pH 5.0 - 6.0 relative to the first and second phases;

- agitation 500 - 1500 rpm; aeration 0.1 - 1.0 litres of oxygen/ litres of culture medium/ minute.

12) The process of claims from 6 to 11 , wherein the value of the pH 5.0 - 6.0 is kept constant during development of the first and second phases by inserting phosphoric acid and ammonia, or phosphoric acid and potassium hydroxide, into the fermenter.

13) The process of claims from 6 to 12, wherein the culture medium has the following composition:

- Phosphoric acid 85%, 26.7 ml; - Calcium sulphate 0.93 g.

Potassium sulphate 18.2 g.

Magnesium sulphate 7H2O 14.9 g. Potassium hydroxide 4.13 g.

- glycerol 40 g. - water 1 litre saline solution 4.35 ml / litre of the above components.

14) The process of claim 7 or 10 or 13, wherein the saline solution has the following composition:

- copper sulphate 5H2O 6.0 g. - Sodium iodide 0.08 g. - manganese sulphate H₂O 3.0 g.

- sodium molybdate 2H₂O 0.2 g.

- boric acid 0.02 g.

- cobalt chloride 0.5g. - zinc chloride 20.Og.

- iron sulphate 7 H₂O 65.Og.

- biotin 0.2g.

- sulphuric acid 5ml.

- water up to one litre 15) The process of claim 7 or 10, wherein the portion of the saline solution is about 12 ml/litre.

16) The process of claims from 6 to 15, wherein at the end of the second phase or the start of the third phase the pH of the culture broth is brought to about 3.0.

17). Recombinant pig lactoferrin obtainable with the processes of claims from 5 to 14.

18) Feed or integrator for animals containing Pichia pastoris RLF9.

19) Feed or integrator for animals containing pig lactoferrin as in claim 16.