WO2009076737A1

WO2009076737A1 - Production of recombinant bos taurus growth hormone with pichia pastoris

Info

Publication number: WO2009076737A1
Application number: PCT/BR2008/000385
Authority: WO
Inventors: Suzana Neiva Santos; Angela Souza Da Fonseca Ramos; Marcus De Melo Teixeira; Lídia Maria PEPE DE MORAES; Dolivar Coraucci Neto; Ricardo Titze De Almeida; Fernando ARARIPE GONÇALVES TORRES; Maria Sueli Soares Felipe
Original assignee: Ouro Fino Participações E Empreendimentos S/A.; Dnatech Projetos Tecnológicos Ltda.,; Fundação Universidade De Brasília
Priority date: 2007-12-14
Filing date: 2008-12-12
Publication date: 2009-06-25
Also published as: CA2746905A1; BRPI0706146A2

Abstract

The present invention provides the production of recombinant bovine growth hormone (rbGH) using a synthetic gene sequence and maintaining the characteristics of natural bovine growth hormone. According to the present invention, is used a synthetic gene sequence that uses preferential codons and optimized content of nucleotide GC bases for P. pastoris. The present process enables the production of this hormone on a large scale and with high efficiency expression.

Description

Production of recombinant bos taurus growth hormone with pichia pastoris

Description

Technical Field

The present invention relates to the production of recombinant bovine {Bos taurus taurus) growth hormone (GH) using a genetic sequence of GH adapted for preferential codons (codon usage) from Pichia pastoris.

More specifically, this invention provides a new gene coding sequence for bovine growth hormone with the codon usage of P. pastoris. This sequence is expressed in yeast P. pastoris, that secrets recombinant bovine growth hormone (rbGH) of protein sequence identical to native to the culture medium.

This invention also includes a manufacturing process of recombinant bovine growth hormone using heterologous expression aforementioned in order to obtain a protein with high productivity, high efficiency and low costs.

Background and Prior Art

The bovine growth hormone (bGH), also called bovine somatotropin, consists of a single polypeptide of 191 amino acid residues, synthesized and secreted by the pituitary gland or anterior pituitary, under the control of the hypothalamus. The bovine somatotropin is responsible for several complex physiological processes, such as growth, metabolism, proteins synthesis, cell proliferation (Kostya, JL & Isalsson, O. lnt Rev Physiol, 13:255-274, 1977), and one of the greatest importance to the agribusiness world, which is the production of milk.

The use of GH is based on its own biological activity described for animals, acting on skeletal growth, weight gain, retention of nitrogen, protein synthesis and in the metabolism of glucose and lipids. The bGH can be given to calves in order to further improve its growth rate and weight gain, reducing the time required between age and time of slaughter, resulting in an increased production of meat. In addition, the bGH can be given to sheep, pigs and other animals used for the same purpose. The administration of bGH in dairy cows is held in various countries to increase the volume of milk production for at least 15%, resulting in significant increase of the producer profitability. In addition, because it is a protein, is not requested a grace period for the consumption of milk treated since the hormone has no effect when taken orally. Brazil, for example, had about 185 million heads of cattle in 2002, which shows a potential consumer market of rbGH. Currently, in Brazil, this protein is made available under the trade names "Lactotropin injectable" by EIIi Lilly do Brasil Ltda., and "Boostin" by Coopers do Brasil Ltda.

The GH, together with another hormone, the prolactin, controls the production of milk during lactogenesis and galactopoiesis, and is essential for the transition from the mammary gland proliferation and the nursing form. In ruminants, the GH is dominant in relation to prolactin in the stage of galactopoiesis, contrary to what occurs in rodents and humans. The gradual decline of the mammary glands at the end of the period of lactation is related to the decrease in the levels of GH, prolactin and IGF-1 , which shows its important role in bovine lactation.

Traditional methods of obtaining growth hormone for use in animals comprise the hormone extraction from the bovine pituitary, recombinant expression in Escherichia coli (Itakura, K. et al. Science, 198:1056-1063, 1977), expression in yeast Saccharomyces cerevisiae cells or expression of recombinants in cell culture (Larrick, J. W. & Thomas, D.W. Curr Opin Biotechnol, 12:411-418, 2001). Strategies for the heterologous expression in organisms are currently more concerned to produce recombinant products. Both transgenic plants and animals, as plants tissues culture have been used to produce a variety of different recombinant proteins (Doran, P. Curr Opin Biotechnol, 11 :199-204, 2000; Larrick, J.W. & Thomas, D.W. Curr Opin Biotechnol, 12:411-418, 2001).

The choice of an expression system for producing high concentrations of recombinant protein depends on many factors, such as characteristics of cell growth, expression levels, intracellular/extracellular expression, post- translational changes, biological activity of the protein of interest and cost of the process (Baneyx, F. Curr Opin Biotechnol, 10:411-421 , 1999). The scheme which uses the bacterium E. coli has shown a high yield of recombinant proteins (Das, A. Methods Enzymol, 182:93-112, 1990; Hewitt, L.. & McDonnell, J. M. Methods MoI Biol, 278:1-16, 2004).

However, despite all the knowledge of genetics and molecular biology from E. coli, many genes are not efficiently expressed in this organism. Among the factors that affect the heterologous expression in E. coli, it is highlighted the composition of nucleotides of the gene (codon usage), the stability and efficiency of translation of mRNA, the folding of proteins, the degradation of the recombinant protein by proteases of the host cell, and toxicity of recombinant protein. In addition, E. coli presents a number of constraints that hinder the heterologous expression, for example, the inability to perform post-translational changes found in eukaryotes proteins, the lack of an efficient secretion mechanism of protein to the culture medium and limited skill for disulphyde bridges formation (Duilio, A. et al. Methods MoI Biol, 267:225-238, 2004). Due to the difficulties encountered in the production of certain eukaryotic proteins in E. coli, other expression systems have been developed.

Cultures of mammals and insects cells, and yeast (fungus), have been used as eukaryotic systems of alternative expression to E. coli. The yeasts have received broad attention in recent years, and offer some advantages of the prokaryotic system, such as simple genetic manipulation and rapid growth. The yeast organisms are able to make post-translational protein modifications typical of eukaryotic cells. Among those employed as expression systems of interest are 5. cerevisiae and P. pastoris, and the latter has received wide acceptance for the production of biopharmaceuticals, since it is capable of doing some post-translational process, such as glycosylation, so very similar to mammals. In addition, P. pastoris allows the extracellular or intracellular production of recombinant protein when grown at high cell densities in simple defined medium (Xiong, A.S. et al. Appl Microbiol Biotechnol 72:1039-47, 2006; Gellissen, G. Appl Microbiol Biotechnol, 54:741-750, 2000). Still, one of the most important features of the yeast P. pastoris compared with S. cerevisiae is its ability to grow in culture medium containing methanol as the sole source of carbon, which is a methylotrophic characteristic. The first step in the use of methanol is the oxidation of this, leading to the formation of formaldehyde and hydrogen peroxide, a reaction catalyzed by the enzyme alcohol oxidase. P. pastoris has two genes that encode for alcohol oxidase, AOX1 and AOX2, but only the former is heavily regulated by methanol while the second is expressed in low quantities. When P. pastoris grows in the presence of glycerol, glucose or ethanol, the alcohol oxidase enzyme is not found, however, in the presence of methanol, the alcohol oxidase enzyme 1 may reach 35% of total cellular proteins. The control of AOX1 gene expression occurs at transcriptional level (Sauer, M. et al. Microbial Cell Factories, 3:17-31 , 2004). The promoter of the gene AOX1 has been widely used for the construction of expression vectors of P. pastoris for being strong and highly adjustable, which reduces the possibility of toxic proteins harming the cell growth. These vectors are integrative type, allowing the stabilization of the exogenous gene message in the genome of yeast.

The expression of recombinant proteins in P. pastoris has shown high levels of production. The system of fed-batch to produce an insulin precursor, produced around 1.5 g/L (Wang, Y. et al. Biotechnol Bioeng, 73:74-79, 2001). The production of dog growth hormone reached 40 μg/mL (Ascacio-Martinez, J.A. & Barrera-Saldana, H.A.. Gene, 340:261-266, 2004), and pig, about 900 mg/L (Ouyang, J. et al. Prot Express Purify, 32:28-34, 2003). For bovine GH, the expression of recombinant protein by heterologous expression system using P. pastoris was described, but with low production using the native gene (Gallardo, HL et al. Uanl Science, 6:339-343, 2003).

Note, however, that there is no sequence of recombinant bovine growth hormone described in the prior art that uses preferential codons for the yeast P. pastoris.

Summary of the Invention Thus, is the main objective of this invention to provide the recombinant bovine growth hormone production in high levels in yeast P. pastoris, using the expression of a synthetic version of the gene that encodes bovine GH.

Another objective of the present invention is to provide an increase in the efficiency of exogenous gene expression, by a chemically synthesized sequence using the preferred codons of P. pastoris.

It is yet another objective of the present invention to provide a process for producing recombinant bovine growth hormone on a large scale and low cost.

Brief Description of the Figures

The attached Figures 1-6 illustrate the results of experiments performed at each stage of bovine growth hormone production in accordance with the examples that follow. These figures show:

Figure 1 - Electrophoresis to confirm the digestion of pBluescript-bGH construct and the release of the bGH insert. Profile on 0.8% agarose gel of the pBluescript-bGH construct digestion and the release of the insert bGH. 1 : intact pBluescript-bGH construct (600ng), 2: pBluescript-bGH construct digested with Xhol / Xbal; M: 1 Kb ladder molecular marker (Promega, Madison Wl₁ USA). The reaction of digestion released the pBluescript-KS vector with approximately 3,500bp and the insert bGH with around 600 bp (bp: base-pairs).

Figure 2 - Electrophoresis to confirm the inclusion of bGH insert into vector pPICZαA. Profile on 0.8% agarose gel of pPICZαA-bGH construct digestion to confirm the inclusion of the bGH insert. M: 1 Kb ladder molecular marker (Promega, Madison Wl₁ USA); 1 : intact pPICZαA-bGH construct; 2: pPICZαA-bGH construct digested with Xhol / Xbal. The reaction of digestion released the vector pPICZαA with 3,600 bp and the bGH insert with around 600bp (bp: base-pairs).

Figure 3 - Immunodetection of recombinant bovine growth hormone (rbGH) expressed by 65 selected clones in 96-hour induction (+), commercial bovine growth hormone; 1-65: selected clones. Figure 4 - Profile of polyacrylamide gel of bovine recombinant growth hormone (bGH) expression in Pichia pastoris in 1L finned bottles with induction for 96 hours. M: molecular weight marker for proteins (Invitrogen, Carlsbad CA); C: commercial bovine growth hormone (Coopers do Brasil Ltda.); Naked: transformed clone with the pPICZαA vector without bGH insert; 3, 4, 18, 31 , 58, 60: selected clones for the expression in 1 L finned bottles. The band referent to the recombinant bovine growth hormone is in the expected height molecular weight of 22 kDa.

Figure 5 - Western Blot of the clones grown in 1L finned bottles for the confirmation of expression of the recombinant bovine growth hormone (rbGH). M: molecular weight marker for proteins (Invitrogen, Carlsbad, CA, USA); C: commercial bovine growth hormone (positive control; Coopers do Brasil Ltda.); Naked: transformed clone with the pPICZαA vector without bGH insert; 3, 4, 18, 31 , 58, 60: selected clones and induced in 1L finned bottles for the production of recombinant bovine growth hormone. The mark in the 22 kDa height indicates that the protein expressed reacts immunologically with the anti-bGH, strongly indicating that the expressed protein is the recombinant bGH.

Figures 6 A and B - Evaluation of the production of recombinant growth hormone (rbGH) by gel densitometry. A: polyacrylamide gel showing the clone band profiles; 31 , 58, 60, Naked: transformed clones grown and induced in 1 L bottle for assessing the recombinant hormone production; C: commercial growth hormone (positive control; Coopers do Brasil Ltda.); B: graphic illustration of the expression efficiency of each clones compared with the commercial growth hormone. %: band expression referring to the bGH compared with other bands of the clone; 18, 31 , 58, 60: transformed clones; +C: commercial growth hormone (Coopers do Brasil Ltda).

Detailed Description of the Invention

The cited objectives are achieved, according to the present invention, by the expression of a synthesized gene sequence of bovine growth hormone in P. pastoris yeast using the preferential codons P. pastoris. This yeast is deposited at the National Center for Agricultural Utilization Research/ARS Patent Culture Collection (USA) under number NRRL Y-50177, 09/24/2008.

The rbGH, according to the present invention, is produced using the yeast P. pastoris, which process differs substantially from that produced using the expression system in E. coli, as revealed in the documents U.S. 5,366,876 (1994), Cho et al.; U.S. 6,229,003 (2001), Aviv et al.; and U.S. 6,692,941 (2004), Miller, et al.. In addition, the gene sequence used for the recombinant protein expression in E. coli usually does not suffer any kind of change. In the process of this invention, the sequence is changed, using preferential codons of P. pastoris.

The nucleotides sequences used as a basis for the design and synthesis of new sequence in accordance with the present invention are described in the public domain database GenBank, accessible at www.ncbi.nih.gov/entrez under the gi codes: 2168581 ; 2168481 ; and 2168363.

The coding sequences of Bos taurus taurus mature polypeptide were analyzed for nucleotides sequences similarity and amino acid residue through Clustal W software, accessible on http://clustalw.genome.jp, as described in biological sequence listing (LSB) LSB 1 and LSB 2, respectively. During analysis was not taken into account the sequences referent to secretion signal, peptide-signal, since this sequence is provided by the pPICZαA expression vector of P. pastoris. Relying on the result of comparison of the sequences above, the selected sequence was described in LSB 3 as reference to the synthesis of new gene, taking into account the presence of amino acid valine at position 127 that, in previous studies, has shown to be the differential in milk production (Eppard, P.J. et al. J Endocrinol; 132:47-56, 1992) (LSB 5). Changes to facilitate the procedures for cloning and to enable the increase of hormone production were performed as described below.

Initially, restriction sites were included in the sequence at both ends to facilitate the cloning process in the expression vector of P. pastoris. At the end 5' has been added a nucleotide sequence related to Xhol restriction site, and at the 3' end, the nucleotide sequence related to Xbal restriction site, as shown in LSB 4. In addition to the restriction sites, a nucleotide sequence was added upstream the GH gene encoding for 6 amino acids residues (L-R-E-A-E-A: Lysine-Arginine-Glutamate-Alanine-Glutamate-Alanine) that determines the site of cleavage by proteases encoded by kex2 and ste 13 genes that are involved in post-translational process and direction of the expressed protein (LSB 4) to the extracellular medium. In the downstream region, after the growth hormone gene sequence, were added nucleotides that encode a stop codon (LSB 4). After above cited alterations, the sequence created has been adapted for preferential codons of the P. pastoris yeast and content of nucleotide GC bases was adjusted to 46.8% (LSB 4) in order to facilitate the stability of the exogenous genetic message integrated into the yeast genome and optimize the translation of its mRNA. The gene is chemically synthesized and cloned into pBluescript KS vector.

With the nucleotide sequence properly cloned into pBluescript KS vector, this construct, called pBluescript-bGH, was inserted into competent cells of E. co/i strain DH5α by heat shock transformation. A maxi-preparation of the plasmid was performed, and then, the release of the bGH insert was processed. The process of release the insert was carried out by digestion with restriction enzymes related to the restriction sites placed in the synthesized sequence, Xhol and Xbal. The digestion reaction generated a fragment with approximately 3',!50O base pairs (bp), referent to the pBluescript KS linearized vector, and a fragment with 607 bp, referent to the synthetic bGH gene, as shown in Figure 1.

The bGH fragment of 607 bp obtained by digestion of the pBluescript- bGH construct was cloned into pPICZαA expression vector (Invitrogen, San Diego, CA, USA) for P. pastoris using the same restriction sites for both vector open site as the insert inclusion site. The new construct, pPICZαA-bGH, was introduced by heat shock transformation in E. co/i strain DH5α and held a maxi- preparation of the vector. The construct was digested with Xhol / Xbal for certification of cloning of bGH insert into the pPICZαA vector, as illustrated in Figure 2. Once confirmed the insert inclusion, the yeast P. pastoris strain X33 (Invitrogen, San Diego, CA, USA) was transformed by electroporation and plated in YPD medium (1 % of yeast extract; 2% peptone; 2% dextrose), supplemented with Zeocina® to select transformed clones, since the pPICZαA vector have the gene for antibiotics resistance. 65 clones were selected, which have undergone through hormone production test. For the assurance that the clones were transformed with the pPICZαA-bGH construct, was performed a PCR - Polymerase Chain Reaction - of colony using oligonucleotides 3¹AOX and 5¹AOX with which the bGH fragment was amplified.

Initially, the 65 clones were grown in 6 ml_ of specific medium in test tubes of 65 ml_ under agitation of 250 rpm in shaker at 30 ⁰C for 96 hours, gathering up aliquots every 24 hours. The presence of bGH in the supernatant was assessed by dot-blotting, as illustrated in Figure 3. From the 65 obtained clones, 6 presented the best productions in the strategy cited above, which were submitted to grow in 1 L finned bottles. The clones were qualitatively evaluated for the production of bGH by SDS-PAGE, illustrated in Figure 4 and western- blot, illustrated in Figure 5. They were quantitatively evaluated by quantification of total protein (Bradford, M. M. Anal Biochem, 72:248-54, 1976) and by gel densitometry, illustrated in Figures 6 A and B.

The clone of best production by the process of the present invention described above, was the number 31 , showing 29.39 mg/L of bGH and efficiency expression of 72.4% in the culture of P. pastoris yeast.

The production process of recombinant bGH using P. pastoris yeast as expression system, according to the present invention, comprises the following basic steps: a) synthesis of the encoding cDNA of bovine growth hormone (Bos taurus taurus); b) adaptation of the synthesized sequence of the cited hormone for preferential codons of the P. pastoris yeast and optimization of the content of nucleotide GC bases; c) inclusion of the restriction site Xhol at the 5' end, and Xbal at the 3' end; d) addition of nucleotides sequences that encode 6 amino acid residues at the 5¹ end immediately upstream the coding bGH sequence, and addition of a stop-codon downstream the sequence; e) cloning of the synthesized gene into expression/secretion vector using methods of molecular biology; f) yeast cell culture in finned bottles for expression of growth hormone; g) evaluation of growth hormone expression; h) quantification of the efficiency expression of recombinant growth hormone.

The following examples illustrate a preferred form of process execution, according to the present invention, without, however, providing a straitened or restrictive character.

Examples

Example 1 - Gene synthesis

The protein sequence of bGH hormone to be produced in P. pastoris was set up after the alignment of various sequences deposited in GenBank, gi: 2168581 ; 2168481 ; 2168363. The sequence defined here does not have secretion signs. For the design of the synthetic gene corresponding to bGH, a table of codon usage of the P. pastoris yeast obtained on the website http://www.kazusa.or.jp/codon/ was used. After the reverse translation of the protein sequence of bGH hormone using the preferential codons of P. pastoris, the sequence obtained was manually altered to change a few nucleotides in order to meet the two criteria: (i) elimination of the restriction sites also present in the pPICZαA expression vector, and (ii) adjustment of the cytosine and guanine content to 46.8%. The synthetic gene was synthesized by Epoch Biolabs (USA), and then cloned into pBluescript KS vector, thus resulting the pBluescript-bGH construct. Example 2 - Transformation of E. coli cells with pBluescript-bGH construct

Were added to 50 μl_ of competent cells from E. coli (DH5α), 1 μL of pBluescript-bGH construct and 9 μL of distilled water. The solution was incubated on ice for 30 minutes, followed by heat shock to bacteria transformation for 90 seconds at 42 ⁰C. Then, 900 μL of LB medium (Tryptone 1%, NaCI 0.5%, yeast extract 0.5%) were added and culture was incubated in a water bath at 37 ⁰C for 1 hour. After incubation, 150 μL of this system were plated in LB agar medium with ampicilin (100 μg/mL) followed by incubation at 37 ⁰C for up to 16 hours. After the clones selection, a maxi-preparation of plasmids was held on.

Example 3 - Protocol of plasmids maxi-preparation A single bacterial clone was inoculated into 5 mL of LB medium containing ampicillin (100 μg/mL) and incubated under agitation of 250 rpm at 37 °C for 16 hours. 1 mL of these pre-culture was inoculated in 250 mL of LB medium with ampicillin (100 μg/mL), followed by incubation at 37 °C under agitation of 250 rpm for 18 hours. The cells were collected by centrifugation at 10,000 x g for 15 minutes at 4 ⁰C, and the supernatant discharged and the pellet resuspended in 17 mL of TE (Tris-HCI [pH 8] 1OmM; EDTA 0.1 mM). The system was incubated on ice for 5 minutes. Next, 34 mL of solution Il were added (NaOH 0.2 M; SDS 1%) prepared on time and the system was carefully mixed by inversion. 25.5 mL of solution III (sodium acetate 3 M; 2 M acetic acid) were added, the tube was inverted several times and the system was centrifuged at 4,000 x g for 30 minutes at 4 ⁰C. The supernatant was transferred to Corex 30 tubes and added 1 volume of isopropanol 100%. The tube was inverted several times and centrifuged to 12,000 x g for 15 minutes at 4 "C.The supernatant was discharged and the pellet resuspended in 17 mL of TE. 9 mL of 7.5 M ammonium acetate were added to the pellet and mixed up vigorously. The solution was centrifuged at 12,000 x g for 15 minutes at 4 ⁰C and the supernatant transferred to a new tube. Were added 62.5 mL of ethanol 100% and tube was inverted several times to mix the phases. The tube was centrifuged at 12,000 x g for 10 minutes at 4 ⁰C, and the supernatant discharged. The pellet was washed with 41 ml_ of ethanol 70%. The tube was centrifuged at 12,000 x g for 15 minutes; and the supernatant was discharged and dried at room temperature. The pellet was resuspended in 100 μl_ of MiIIiQ water plus 4 μl of 20 mg/mL RNAse solution.

Example 4 - Digestion reaction of pBluescript-bGH construct, inserts release and band retrieval by gel elution

For a digestion reaction of 30 μl_ of the final volume, were used 2 μg of pBluescript-bGH construct, 3.0 μl_ of 10X buffer (Invitrogen, San Diego CA, USA; buffer 2), 2.0 μl_ of Xhol (Invitrogen, San Diego CA, USA; 10 U/μL), 2.0 μL of Xbal (Invitrogen, San Diego CA, USA; 10 U/μL) and distilled water sufficient for the final volume of the reaction. The reaction was incubated at 37 ⁰C for 10 hours. After incubation, the result of digestion was evaluated by 0.8% agarose gel electrophoresis and the digestion pattern displayed, as illustrated in Figure 1.

The insert of 607 bp released by the digestion reaction was eluted in the agarose gel using the protocol described in manuals of commercial kit WizardR SV Gel and PCR Clean-Up System - Promega (Madison Wl, USA).

Example 5 - Cloning of bGH synthetic nucleotide seguence in pPICZαA vector

For the process of bGH insert sub-cloning into pPCICZαA expression vector, initially it was necessary a vector linearization. Were used 2.5 g of pPICZαA vector, obtained from maxi-preparation of plasmids, 4.5 μL of 10X buffer (Invitrogen, San Diego CA, USA; buffer 2), 2.0 μL of Xhol (Invitrogen, San Diego CA, USA; 10 U/μl), 2.0 μL of Xbal (Invitrogen, San Diego CA, USA; 10 U/μL) and enough water to a final reaction volume of 45 μL. The digestion reaction was incubated at 37 ° C for 10 hours.

With the linearized vector, the binding of bGH insert into pPICZαA vector was realized. Were used 100 ng of vector, 50 ng of bGH insert, 2 μL of T4 ligase buffer (Promega, Madison Wl, USA), 1.0 μL of T4 DNA ligase (Promega, Madison Wl, USA; 3 U/μL) in final reaction volume of 10 μL. The reaction was incubated at 16 ⁰C for 14 hours. The system was then used for the transformation of E. coli by heat shock, as previously described. The plasmids maxi-preparation derived from a transformed clone was performed followed by digestion with restriction enzymes for the certification of the sub-cloning, as previously mentioned and illustrated in Figure 2. The plasmid obtained was called pPICZαA-bGH.

Example 6 - Transformation of P. pastoris cells, strain X33. with pPICZαA-bGH construct

In order to obtain competent yeast cells for transformation, it was necessary the growth of the X33 strain (Invitrogen, San Diego CA, USA) in 5 mL of YPD (yeast extract 1%; peptone 2%; dextrose 2%) in a 125 mL Erlenmeyer flask, at 30 ⁰C for 16 hours. After the growth period, were inoculated 500 μl_ of culture in 100 mL of YPD medium. The culture was incubated at 30 ⁰C for 16 hours until an OD600 of 1 ,3 - 1 ,5. The cells were collected by centrifugation at 1 ,500 x g for 5 minutes at 4 ⁰C. The pellet was resuspended in 100 mL of ice sterile water followed by a new centrifugation at 1 ,500 x g for 5 minutes at 4 ^CC, and the pellet was resuspended in 50 mL of ice sterile water. On the new pellet were added 10 mL of ice sterile 1 M sorbitol. The cells were collected by centrifugation at 1 ,500 x g for 5 minutes at 4 ⁰C and the pellet was resuspended in 1 mL of ice sterile 1 M sorbitol, up to a final volume of 1.5 mL. The cells were sedimentated by centrifugation at 1500 x g for 5 minutes at 4 ⁰C and the pellet was resuspended in 1 mL of ice sterile 1 M sorbitol, up to a final volume of 1.5 mL. Then, 5 to 10 μg of linearized DNA in 5 - 10 μL of water were mixed to 80 μL of cells and the system was transferred to an ice electroporation bucket of 0.2 cm, followed by incubation for 5 minutes on ice. The cells were subjected to electroporation under the following conditions: 1500 V, 400 Ω, 25 μF and time of electroporation close to 10 ms with a field of 7500 V.cm^"1. About 200 μL of the transformation system were plated in YPDS medium (yeast extract 1%; peptone 2%; dextrose 2%; sorbitol 1 M; agar 2%) plus Zeocina® 100 μg/mL, and the plates were incubated at 30 ⁰C for 4 days for colonies growth. Example 7 - Yeast cell culture in 1 L finned bottles for GH expression

Once the producer's clones were selected, fermentation tests were performed in 1L bottles, toward a greater production of rbGH. A single colony was inoculated in 25 ml of BMGY medium (yeast extract 1%, peptone 2%; 10OmM potassium phosphate buffer, pH 6.0; YNB 1.34%; biotin 4 x 10-5%, glycerol 1%) in a 250 ml_ finned bottle. The culture was incubated at 28-30 ⁰C in an incubator at 250 rpm until the culture reached OD600 of 2-6, about 18 hours. The cells were collected by centrifugation at 3,000 x g for 5 minutes at room temperature. The supernatant was discharged and the pellet was washed with 10 ml_ of sterile distilled water. The culture was centrifuged at 3,000 x g for 5 minutes at room temperature. The precipitate was resuspended in 100 ml_ of BMMY medium (yeast extract 1%, peptone 2%; 10OmM potassium phosphate buffer, pH 6.0; YNB 1.34%; biotin 4 x 10-5%, methanol 0.5%) to a final OD600 of 1 to 2. The induction was held in a 1 L finned bottle, cover with wad of gauze and cotton to facilitate aeration culture. The culture was incubated in a shaker at 250 rpm for 96 hours. After the first 24 hours, 100% methanol was added to culture to a final concentration of 0.5% and thus maintained every 12 hours. 5 ml_ aliquots were gathered up every 24 hours for analysis. As bGH expression in X33 strain is extracellular, the aliquots collected were centrifuged to cell sedimentation at 3,000 x g for 5 minutes. The supernatant was subjected to the procedure of proteins precipitation for analysis by SDS-PAGE.

Example 8 - Precipitation of expressed proteins in the samples collected during fermentation and evaluation by SDS-PAGE

From 1 ml_ sample of the supernatant properly centrifuged, were added 250 μl_ of 100% TCA (trichloroacetic acid, Sambrook et al., 1989) and incubated at -20 ⁰C for 1 hour. The system was centrifuged at 12,000 x g for 15 minutes at 4 ⁰C. The supernatant was discharged and the pellet washed with 500 μl_ of 100% ice acetone. The tube was centrifuged to 12,000 x g for 10 minutes at 4 ⁰C. The supernatant was discharged and washed twice. The pellet was dried at room temperature for 2 hours or in oven at 45 ⁰C for 20 minutes. The precipitate was resuspended in 100 μl_ of buffer sample 1X and, when necessary, the pH of the sample was adjusted by adding 3M Tris-HCI pH 8.8. To evaluate the fermentation process, the samples were incubated in boiling water bath for 10 minutes and 25 μl_ applied on a 15% polyacrylamide gel, with 4% gel hub. It was applied a current of 10 mA and 50 V. After the race, the gel was stained with Coomasie blue 0.4% and discolored with a discoloring solution of acetic acid 10%, ethanol 43.5%. Figure 5 illustrates this assessment.

Example 9 - Evaluation of the GH expression of by Western Blot.

The western-blot technique was performed using the semi-dry transfer system. After the separation of polypeptides by polyacrylamide gel electrophoresis, the material was transfered to a nitrocellulose membrane (Hybond-C Extra, Amersham Biosciences, UK) with the same size of a polyacrylamide gel. The nitrocellulose membrane, the gel of polyacrylamide, and 12 filter paper in the dimensions of the gel were immersed in transfer buffer of Tris-HCI 48 mM; Glycine 39 mM; SDS 0.037%, Methanol 20%.The transfer system used was the semi-dry with graphite electrodes (Pharmacia-LKB®), where the gel and membrane were between the roles of filter paper equally divided. It was then submitted to an electric current with 0.8 mA.cm^"2 amperage for 2 hours. After the transfer, the membrane was blocked with 1X PBST solution (NaCI 137 mM; Na₂HPO₄ 7mM; NaN₃ 0.02%, tween 20 0.1%) and 5% skimmed milk for 2 hours at room temperature under mild agitation. The membrane was washed with 1X PBST solution and incubated with anti-bGH primary antibody (VMRD Inc., Pullman WA, USA; 1mg/mL) diluted 1 :500 for 2 hours at room temperature under mild agitation. The membrane was washed again with 1X PBST and incubated with conjugated anti-mouse IgG secondary antibody with alkaline phosphatase (Promega, Madison Wl, USA) diluted 1 :5000, for 2 hours at room temperature and under mild agitation. The membrane was washed with 1X PBST solution and APB solution (Tris-HCI 10OmM, NaCI 10OmM; 5mM MgCI₂), and then submitted to the revelation with NBT (Promega, Madison Wl, USA; 50mg/mL) and BCIP (Promega, Madison Wl, USA; 50 mg/mL). Figure 6 illustrates this assessment.

Example 10 - Efficiency of rbGH expression Based on the growth of clones in 1L bottles, aliquots were collected and evaluated for the amount of proteins expressed by the method of Bradford (Bradford, M. M. Anal Biochem, 72:248-54, 1976). Together with gel densitometry analysis performed by the Scion Image software (Scion Corporation, USA) and Origin 7.5 (OriginLab Corporation, USA), the efficiency of the rbGH expression was quantified.

Example 11 - Colony PCR

Based on a small quantity of cells from a transformed colony of P. pastoris, 150 μl_ of MiIIiQ sterile water were needed to dissolve the colony. The content was incubated in boiling water bath for 10 minutes, and from these solution, 4 μL were used as DNA template for PCR reaction including 2.5 μl_ of 10X buffer (Cenbiot Biotechnology, Porto Alegre RS, BR); DNTP 0,25 mM, MgCI2 1.5 mM, Taq DNA polymerase 2U (Cenbiot Biotechnology, Porto Alegre RS₁ BR), oligonucleotides 3¹AOX (5¹GCAAATGGCATTCTGACATCC) and 5¹AOX (5'GACTGGTTCCAATTGACAAGC) 0.4 mM (each); and distilled water sufficient to a 25 μL reaction volume. The reaction was performed in thermal cycler under the following conditions: 94 ⁰C for 3 minutes, 26 cycles of 1 minute at 94 ⁰C, 56 ⁰C for 1 minute and 72 ⁰C for 45 seconds. The final extension was 5 minutes at 72 ⁰C and incubation at 4 ^CC for an indefinite period. The reaction was evaluated on 0.8%. agarose gel.

Biological Sequence Listing (LSB)

LSB 1 : Similarity analysis of the selected nucleotide sequences of Bos taurus taurus from Gen Bank.

Sequence type explicitly set to DNA; Sequence format is Pearson

E00293 (573 bp); E00183 (573 bp); E00057 (573 bp): CLUSTAL W (1.83) multiple sequence alignment

E00293GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGC TGTGCTCCGGGCTCAGCAC

E00183GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGC TGTGCTCCGGGCTCAGCAC E00057GCCTTCCCAGCCATGTCCTTGTCCGGCCTGTTTGCCAACGC

TGTGCTCCGGGCTCAGCAC

_{************************************************************}

E00293CTGCATCAGCTGGCTGCTGACACCTTCAAAGAGTTTGAGCG CACCTACATCCCGGAGGGA

EOO 183CTGCACCAGCTGGCTGCTGACACCTTCAAAGAGTTTGAGCG CACCTACATCCCGGAGGGA

E00057CTCCACCAGCTGGCTGCTGACACCTTCAAAGAGTTTGAGCG

TACCTACATCCCCGAGGGA

_{** ** *********************************** *********** ******}

E00293CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTT CTCTGAAACCATCCCGGCC

EOO 183CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTT CTCTGAAACCATCCCGGCC

E00057CAGAGATACTCCATCCAGAACACCCAGGTTGCCTTCTGCTT

CTCCGAAACCATCCCGGCC

_{******************************************** ***************}

E00293CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGG AGCTGCTTCGCATCTCACTG

EOOIδSCCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGG AGCTGCTTCGCATCTCACTG

E00057CCCACGGGCAAGAATGAGGCCCAGCAGAAATCAGACTTGG AGCTGCTTCGCATCTCACTG

_{****************************}

E00293CTCCTCATCCAGTCGTGGCTTGGGCCCCTGCAGTTCCTCAG CAGAGTCTTCACCAACAGC

E00183CTCCTCATCCAGTCGTGGCTTGGGCCCCTGCAGTTTCTCAG CAGAGTCTTCACCAACAGC

E00057CTCCTCATCCAGTCGTGGCTTGGGCCCCTGCAGTTTCTCAG

CAGAGTCTTCACCAACAGC

_{*********************************** ************************} E00293TTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAA GGACCTGGAGGAAGGCATC

E00183TTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAA GGACCTGGAGGAAGGCATC

E00057TTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAA

GGACCTGGAGGAAGGCATC

_{************************************************************}

E00293CTGGCCCTGATGCGGGAGGTGGAAGATGGCACCCCCCGGG CTGGGCAGATCCTCAAGCAG

E00183TTGGCCCTGATGCGGGAGGTGGAAGATGGCACCCCCCGGG CTGGGCAGATCCTCAAGCAG

E00057πGGCCCTGATGCGGGAGCTGGAAGATGGCACCCCCCGGG

CTGGGCAGATCCTCAAGCAG

_{***************** *****************************************}

E00293ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGC GCTGCTCAAGAACTACGGT

EO0183ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGC GCTGCTCAAGAACTACGGT

E00057 ACCTATGACAAATTTGACACAAACATGCGCAGTGACGACGC

GCTGCTCAAGAACTACGGT

_{************************************************************}

E00293CTGCTCTCCTGCTTCCGGAAGGACCTGCATAAGACGGAGAC GTACCTGAGGGTCATGAAG

EOO 183CTGCTCTCCTGCTTCCGGAAGGACCTGCATAAGACGGAGAC GTACCTGAGGGTCATGAAG

EOOOδTCTGCTCTCCTGCTTCCGGAAGGACCTGCATAAGACCGAGAC

GTACCTGAGGGTCATGAAG

_{*********************************** ************************}

E00293TGCCGCCGCTTCGGGGAGGCCAGCTGTGCCTTC E00183TGCCGCCGCTTCGGGGAGGCCAGCTGTGCCTTC E00057TGCCGCCGCTTCGGGGAGGCCAGCTGTGCCTTC *********************************

LSB 2: Similarity analysis of the selected amino acids sequences of Bos taurus taurusixom Gen Bank.

Sequence type explicitly set to Protein; Sequence format is Pearson

Sequence 1 : E00293191 aa

Sequence 2: E00183191 aa

Sequence 3: E00057191 aa

CLUSTAL W (1.83) multiple sequence alignment

E00293AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRY SIQNTQVAFCFSETIPA

E00183AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRY SIQNTQVAFCFSETIPA

EOOOδTAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRY

SIQNTQVAFCFSETIPA

_{************************************************************}

E00293PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFG TSDRVYEKLKDLEEGI

EOOIβSPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFG TSDRVYEKLKDLEEGI

E00057PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFG TSDRVYEKLKDLEEGI

E00293LALMREVEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLS CFRKDLHKTETYLRVMK

EOOIδSLALMREVEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLS CFRKDLHKTETYLRVMK

E00057LALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLS

CFRKDLHKTETYLRVMK

_******._{*****************************************************}

E00293CRRFGEASCAF E00183CRRFGEASCAF E00057CRRFGEASCAF

_***********

LSB 3: Selected gene sequence for the synthesis of new the gene. gccttcccagccatgtccttgtccggcctgtttgccaacgctgtgctccgggctcagcacctgcatcag ctggctgctgacaccttcaaagagtttgagcgcacctacatcccggagggacagagatactccatccagaac acccaggttgccttctgcttctctgaaaccatcccggcccccacgggcaagaatgaggcccagcagaaatca gacttggagctgcttcgcatctcactgctcctcatccagtcgtggcttgggcccctgcagttcctcagcagagtctt caccaacagcttggtgtttggcacctcggaccgtgtctatgagaagctgaaggacctggaggaaggcatcctg gccctgatgcgggaggtggaagatggcaccccccgggctgggcagatcctcaagcagacctatgacaaatt tgacacaaacatgcgcagtgacgacgcgctgctcaagaactacggtctgctctcctgcttccggaaggacctg cataagacggagacgtacctgagggtcatgaagtgccgccgcttcggggaggccagctgtgccttc

LSB 4. Synthesized gene sequence of bovine growth hormone and deducted sequence.

A

^■ SItio KBQ p* SItio STE13 ϋGAGAGGCTGAAGCTGCCTTCCCAGCTATGTCCCTCTCT

GGTTTGTTCGCTAACGCTGTCTTGCGTGCGCAACATTTGCACCAATTG GCTGCTGACACCTTCAAGGAATTCGAAAGAACCTACATCCCAGAGGGA CAGCGTTACTCTATCCAGAACACTCAAGTTGCTTTCTGCTTCTCCGAG ACTATCCCAGCTCCAACTGGAAAAAACGAAGCCCAACAGAAGTCTGAC TTGGAGTTGCTCAGAATCTCTTTGTTGTTGATTCAGTCTTGGTTGGGT

CCATTGCAATTCTTGTCCAGAGTTTTCACCAACTCTTTGGTTTTCGGT ' ^I"⁶"^{013 BGH}

ACTTCCGACCGTGTCTACGAAAAGTTGAAGGACTTGGAAGAAGGTATC

CTCGCTTTGATGAGAGAGGTTGAGGACGGTACCCCTAGAGCCGGTCAA

ATTTTGAAGCAAACCTACGACAAGTTCGATACCAACATGAGATCTGAC

GACGCTTTGTTGAAGAACTACGGTTTGTTGTCTTGTTTCAGAAAGGAT

TTGCACAAGACTGAGACTTACTTGCGTGTCATGAAGTGTAGAAGATTC

B r Sitio KEX2 I — ► Sftio STE13

LEϋEAElAFPAMS LSGLFANAVLR

AQHLHQLAADTFKEFERTYIPEGQR

YSIQNTQVAFCFSETIPAPTGKNEAQ

QKSDLELLRISLLLIQSWLGPLQFLSR

VFTNSLVFGTSDRVYEKLKDLEEGIL

ALMREVEDGTPRAGQILKQTYDKFD

TNMRSDDALLKNYGLLSCFRKDLHK

TETY LRVMKCRRFGEASCAF-

LSB 5. Alignment of sequences used as a reference for the synthesis of the bGH gene and the synthetic bGH gene. The valine residue at position 127 is in focus.

CLUSTAL W (1.83) multiple sequence alignment

gi_2168581 AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPA 60 gi_2168481 AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPA 60 bGH AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPA 60 gi_2168363 AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPA 60

************************************************************ gi_2168581 PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGI 120 gi_2168481 PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGI 120 bGH PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGI 120 gi_2168363 PTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGI 120

************************************************************ gi_2168581 LALMREgEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMK 180 gi_2168481 LALMREgIDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMK 180 bGH LALMRE&DGTPRAGQI LKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMK 180 gi_2168363 IALMREgpDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMK 180

**** ** gi_2168581 CRRFGEASCAF- 191 gi_2168481 CRRFGEASCAF- 191 bGH CRRFGEASCAF- 191 gi_2168363 CRRFGEASCAF- 191 ***********

Claims

Claims Production of recombinant BOS TAURUS growth hormone

1. Production of recombinant Bos taurus growth hormone, comprising the use of a synthetic gene sequence adapted to the preferential codons (codon usage) of the Pichia pastoris yeast, maintaining the characteristics of the natural bovine growth hormone.

2. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein the synthesized gene sequence has restriction sites Xhol / Xbal at their ends.

3. Production of recombinant Bos taurus growth hormone according to claim 1 , characterized by an insertion of a nucleotide sequence at the 5' end in the gene sequence encoding for 6 recognition amino acids by processing proteins encoded by kex2anύ ste13 genes.

4. Production of recombinant Bos taurus growth hormone according to claim 1 , characterized by an insertion of a nucleotide sequence at the 3¹ end in the gene sequence that code for a stop codon in the recombinant protein formation.

5. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein the chemical synthesis use as reference the preferred codons of the Pichia pastoris yeast, optimizing the content of nucleotide GC bases for the microorganism.

6. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein is selected the gene sequence that after the translation presents a valine at position 127.

7. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein is present a positive result in tests of the hormone production by dot blotting method.

8. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein detected the presence of the hormone and its quantification, through induction in 1 L bottles, by polyacrylamide gel and western blotting analysis.

9. Production of recombinant Bos taurus growth hormone according to claim 1 , wherein the amount of hormone produced reached around 29.39 mg/L with efficiency expression of about 72.4%.

10. Process for production of recombinant bGH, comprising the following basic steps: a) synthesis of the encoding cDNA of bovine growth hormone (Bos taurus taurus); b) adaptation of the synthesized sequence of the cited hormone for preferred codons of the P. pastoris yeast and optimization of the content of nucleotide GC bases; c) inclusion of the restriction site Xhol at the 5' end, and Xbal at the 3¹ end; d) addition of nucleotides sequences that encode 6 amino acid residues at the 5' end immediately upstream the coding bGH sequence, and addition of a stop codon downstream the sequence; e) cloning of the synthesized gene into expression/secretion vector using methods of molecular biology; f) yeast cell culture in finned bottles for expression of growth hormone; g) evaluation of growth hormone expression; h) quantification of the efficiency expression of recombinant growth hormone.

11. Gene sequence, characterized as defined below:

Seqϋέnda bGH