WO2002031178A1 - An artificial gene and vectors for expressing high-yield recombinant ovine interferon-tau in pichia pastoris - Google Patents

Abstract

Methods and expression vectors encoding for the ovine interferon-tau (OvIFN-tau) protein are provided for high yield production in any industrial yeast, such as Pichia pastoris. Compared to previous studies, significantly higher levels of correctly-folded and biologically active OvIFN-tau polypeptides are produced by using two different Pichia pastoris expression vectors comprising an optimized IFN-tau nucleotide sequence fused to the α-MF pre-pro signal sequence under the control of the Pichia pastoris AOX1 promoter.

Description

AN ARTIFICIAL GENE AND VECTORS FOR EXPRESSING HIGH-YIELD RECOMBINANT OVINE INTERFERON-TAU IN

PICHIA PASTORIS

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the field o-f molecular biology of interferons. More specifically, the present invention relates to the construction of an artificial gene and development of expression vectors and process for optimizing the production of recombinant ovine interferon-tau (rOvIFN-tau) in Pichia pastoris.

Description of the Related Art

Ott et al. (1991) performed the first work on the expression of recombinant ovine interferon-tau (rOvIFN-tau) in yeast. The ovine IFN-tau gene was modified into a synthetic gene allowing further mutagenesis studies. The authors flanked the IFN-tau gene into a Sac char omyces cerevisiae expression vector that consists of the glyceraldehyde phosphate dehydrogenase/alcohol dehydrogenase II promoter (AGp) and the Ubiquitin coding sequence as a signal peptide for secretion of th e protein into the medium. Protein expression in bulk flasks resulted in 100 mg of rOvIFN-tau per liter of Saccharomyces cerevisiae culture.

van Heeke et al. (1996) attempted to express OvIFN- tau in Pichia pastoris using a vector which enabled secretion of the IFN-tau into the culture media. The original coding region of the OvIFN-tau was cloned in a multicopy expression plasmid pHIL-Sl that consists of the alcohol oxidase I (AOX1) promoter flanked by the 5' and 3' AOX1 gene sequence and an acid phosphatase 1 (PHO1) signal sequence to secrete proteins. This vector was supposed to be highly efficient to integrate multicopy of the OvIFN-tau gene into the Pichia pastoris genome. Transformation experiments showed that the construct pMLD3031Stl l integrated two copies of the gene into the genome. However, the clone GS115/#29 with two integrated copies in the genome was as good producer as clones with a single OvIFN-tau gene copy. Overall, protein expression studies in shake flasks and fermentation of the most efficient clone GS 115/#29 demonstrated yields of 50 and 280 mg per liter of culture respectively.

Trying to overcome some of the above disadvantages and still employ the economical Pichia pastoris expression system, Johnson et al. (1999) tried to modify the culture growing conditions of the same Pichia , pastoris clone. For NMR experiments, ¹⁵N-labeled NH₄C1 was used as a sole nitrogen source and 8-10 mg of ¹⁵N rOvIFN-tau per liter of culture was obtained. One of the problems in low protein expression level in

Pichia pastoris is due to inefficient protein maturation, translocation, and secretion into the medium. Once secreted, a n additional problem could be due to the susceptibility to endogenous host proteases. At the polynucleotide level, high efficient expression in Pichia pastoris required modification of th e original gene, but this modification has not been done before.

Another problem in the production of rOvIFN-tau is the fermentation process. Optimization of conditions to get high- cell density is required for high-yield of protein. Such a process was investigated initially by van Heeke et al. (1996). However, only suboptimal fermentation conditions were obtained.

Thus, although a few reports demonstrated expression of rOvIFN-tau in yeast, the expression levels in those reports were not high enough. This has been a problem since a high level economical production of the protein is critically desirable for pre - clinical and clinical trials and for expressing large quantities of 13C/15N-labeled proteins for structural analysis using multidimensional Nuclear Magnetic Resonance (NMR).

Hence, the prior art is deficient in expression constructs and method of high yield production of recombinant interferon-tau in yeast. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The current invention is directed at a more economical and efficient process for producing recombinant ovine interferon- tau in Pichia pastoris. An artificial OvIFN-tau gene w as constructed and cloned into two different Pichia pastoris expression vectors that consist of the alcohol oxidase I (AOX1) promoter flanked by the Sac char omyces cerevisiae α-mating polypeptide (α-MF) signal sequence to secrete these proteins efficiently. Moreover, using the fermentor available, the present invention developed optimized fermentation conditions that allow high-yield production of the protein.

The artificial OvIFN-tau gene was designed to optimize gene expression by (i) reducing repetitive sequences; (ii) lowering overall G+C content; (iii) reducing or eliminating palindromic sequences; and (iv) optimizing the nucleotidic sequence using th e biased codon usage for a particular yeast. According to these criteria, artificial oligonucleotides were designed and an artificial OvIFN-tau gene was constructed by polymerase chain reaction (PCR). The artificial gene was designed to optimize gene expression in Pichia sp. Since other yeast genera or species are genetically related to Pichia sp., optimization of rOvIFN-tau expression in other yeasts requires only minor modifications to the artificial gene disclosed herein. Other related yeast species include Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Yarrowia sp. and Candida sp.

For effective secretion, the Saccharomyces cerevisiae -mating signal sequence polypeptide (α-MF) was preferred to other signal peptides used previously such as Pichia pastoris acid phosphatase signal peptide (PHOl) (van Heeke et al., 1996). The α-MF prepro signal sequence is the classical and most widely used secretion signal. Although variability in the amino terminus m ay occur, it has been used with the most success for m any biologically active heterologous proteins secreted by Pichia pastoris (Cereghino et al., 2000). However, AOX1 promoter w as kept to ensure tight regulation of gene induction.

Superior results were achieved in the present invention when using a Pichia pastoris strain X33 that was not used before according to the knowledge of the present inventors, or strain GS115 as used previously for OvIFN-tau expression (van Heeke et al., 1996). Expression results in shake flask using transformants GS 115/pPIC9K-OvIFN-tau4 and X33/pPICZαA- OvIFN-tau3 of the present invention resulted in protein production of 584 mg/1 and 690 mg/1 respectively. These results represent a 9 to 11 fold increase when compared to the 50 mg/1 obtained by van Heeke et al. (1996). The most efficient transformant, namely X33/pPICZαA-OvIFN-tau3, was used for scale-up experiments. Using this clone, fermentation results demonstrated yields as high as 1350 mg/1. When compared to th e yield of 280 mg/1 obtained by van Heeke et al., this represents a 5-fold production increase.

In one embodiment of the present invention, there is provided an artificial ovine interferon-tau gene designed for high yield protein production in yeast, wherein the gene is constructed by (i) reducing repetitive sequences; (ii) lowering overall GlC content; (iii) reducing or eliminating palindromic sequences; an d (iv) optimizing the nucleotide sequence of the gene using th e biased codon usage in yeast. In general, the artificial gene is applicable as is and further optimized to yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Yarrowia sp., Pichia sp. and Candida sp. More preferably, the recombinant gene encoding for ovine IFN-tau optimized for Pichia sp. has the sequence shown in SEQ ID No. 13 or 15.

In another embodiment of the present invention, there is provided two expression vectors designed for high yield ovine interferon-tau production in Pichia pastoris. These vectors comprise of (a) an artificial ovine interferon-tau gene constructed by (i) reducing repetitive sequences; (ii) lowering overall GfC content; (iii) reducing or eliminating palindromic sequences; and (iv) optimizing the nucleotide sequence of the gene using th e biased codon usage in Pichia pastoris; (b) a Saccharomyces cerevisiae α-mating polypeptide signal sequence for efficient secretion of protein into the media; (c) an inducible alcohol oxidase I promoter; and (d) regulatory sequences effective for expressing said interferon-tau in Pichia pastoris. Preferably, th e artificial ovine interferon-tau gene has the sequence of SEQ LD No. 15. Representative examples of expression vectors constructed according to the present invention include pPICZαA-OvIFN-tau and pPIC9K-OvIFN-tau.

In yet another embodiment of the present invention, there is provided a method for high yield production of ovine interferon-tau in Pichia pastoris, comprising the steps of: transforming said yeast with the expression vector disclosed herein; inducing protein expression with methanol; culturing said yeast in defined culture conditions; and purifying the protein from the culture media. Preferably, the yeast is Pichia pastoris X33 or Pichia pastoris GS115. The yeast may be cultured in a shake flask or a fermentor.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of th e invention. These embodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows the map of the designed ovine interferon-tau gene. Figure 1A depicts a map of the designed OvIFN-tau gene (SEQ ID No: 13) using overlapping oligonucleotides and the deduced amino acid sequence (SEQ ID NO: 16). Figure I B illustrates the restriction map of the synthetic gene OvIFN-tau (SEQ ID No: 14 and 17). Figure IC illustrates the expected amino-termini processing of the recombinant OvIFN-tau by Kex2 and Stel3 Pichia pastoris proteases.

Figure 2 shows the map of expression vectors for OvIFN-tau. Figure 2A depicts the map of the expression vector pPICZαA-OvIFN-tau5. The EcoRI-Notl fragment of the plasmid pPICZαA was replaced by the EcoRI-Notl OvIFN-tau fragment. Figure 2B depicts the map of the expression vector pPIC9K- OvIFN-tau7. The EcoRI-Notl fragment of the plasmid pPIC9K w as replaced by the EcoRI-Notl OvIFN-tau fragment.

Figure 3 shows the nucleotide sequence (SEQ ID NO: 15) of the EcoRI-Notl insert of pPICZ A-OvIFN-tau5 and pPIC9K- OvIFN-tau7. Figure 4 shows 15% Tricine-PAGE of different examples of X33/pPICZαA-OvIFN-tau and GS 115/pPIC9K-OvIFN- tau Pichia pastoris transformants cultured in shake flasks.

Figure 4A depicts a 15% Tricine-PAGE of different

X33/ρPICZαA-OvIFN-tau transformants expressing OvIFN-tau in shake flask culture after 72h of 1% methanol induction in GMMY medium. Lane 1: supernatant from X33 strain transformed with pPICZαA (Control); Lane M: Molecular weight standard; Lanes 2-7 : supernatant from X33 transformants pPICZαA-OvIFN-tau2, -tau3 , -tau4, -tau5, -tau9, and -taul4.

Figure 4B depicts a 15% Tricine-PAGE of different GS 115/pPIC9K-OvIFN-tau Pichia pastoris transformants expressing OvIFN-tau in shake flask culture after 72h of 1% methanol induction in GMMY medium. Lane 1: supernatant from X33 strain transformed with pPIC9K (Control); Lane M: Molecular weight standard; Lanes 2-9: supernatant from GS 1 15 transformants pPIC9K-OvIFN-tau4, -tau6, -tau7, -tau8, -tau9, -taul l , -taul4, -tau23.

Figure 5 shows the double-step chromatographic purification of the transformant X33/pPICZα-OvIFN-tau3. Optical absorbance is indicated. Figure 5 A depicts a part of an elution curve of anion exchange chromatography on Q-Sepharose Fast Flow. Elution was carried out with a linear concentration of Tris- HC1 50 mM - NaCl 0.35 M, pH 7.6. Figure 5B depicts a part of a n elution curve of gel filtration chromatography on HR100 Sephacryl. Elution was carried out with a linear concentration of Tris-HCl 50 mM - NaCl 0.25M, pH 7.6.

Figure 6 shows OvIFN-tau expression of the clones X33/pPICZαA-OvIFN-tau3, GS 115/pPIC9K-OvIFN-tau4, an d

GS 115/#29. Figure 6 A depicts OvIFN-tau expression using clones X33/pPICZαA-OvIFN-tau3 (•), GS 1 15/pPIC9K-OvIFN-tau4 ( ), and GS 115/#29 (A) in shake flask experiments. Figure 6 B depicts a time course of fermentation of X33/pPICZαA-OvIFN- tau3. Optical absorbance (OD595 nm) (■), wet cell pellet (A), and dissolved oxygen (•) were indicated. Figure 6C depicts a 15% Tricine-PAGE from fermentation experiments of X33/pPICZαA- OvIFN-tau3 during a methanol induction time course. Lane M: prestained molecular weight standard; Lane 1 : sample control during the starvation phase ^• and just prior to the 0.4% methanol induction phase; Lane 2: 6 hours after 0.6% methanol induction; Lane 3: 24 hours after 1% methanol induction; Lane 4: 48 hours after 1.5% methanol induction; Lane 5: 72 hours after 1.5% methanol induction; Lane 6: 94 hours after 1 % methanol induction.

Figure 7 shows MS MALDI-TOF spectra of th e purified X33/pPICZαA-OvIFN-tau3 and GS 115/pPIC9K-OvIFN- tau4 from bulk flask, and X33/pPICZαA-OvIFN-tau3 from fermentation experiments. Figure 7 A depicts MALDI-TOF spectrum of the purified OvIFN-tau expressed by X33/pPICZαA- OvIFN-tau3 transformant in shake flask. Figure 7B depicts MALDI-TOF spectrum of the purified OvIFN-tau expressed b y X33/pPICZαA-OvIFN-tau3 transformant in fermentation experiments. Figure 7C depicts MALDI-TOF spectrum of the purified OvIFN-tau expressed by GS 115/pPIC9K-OvIFN-tau4 transformant in shake flask.

Figure 8 shows 1D-NMR spectra of the purified

X33/OvIFN-tau3 from bulk flask (Figure 8A) and fermentation (Figure 8B) experiments.

Figure 9 shows the Circular Dichroism (CD) spectrum of ¹⁵N-labeled OvIFN-tau at 1.5 mg/ml, pH 7.2, Tris-HCl 1 mM, 2 0

C recorded on an Aviv 62DS spectropolarimeter using 0.01 m m cell. Five scans from 260 to 185 nm were averaged using a 2 - mm bandwidth, 1 sec averaging time, and 0.5 mm intervals.

Figure 10 shows antiviral activity of the purified

X33/pPICZαA-OvIFN-tau3 from bulk flask (Figure 10A) and fermentation experiments (Figure 10B) towards MDBK-cell line. Serial dilutions in duplicate were made of the 1 mg/ml X33/pPICZαA-OvIFN-tau3 stocks. One unit of IFN-tau is defined as the reciprocal of the dilution at which 50% protection is observed. In Figure 10A , both stocks have 10⁸U/ml. In Figure 10B , the first stock has 10⁸U/ml, the second stock has 10^{7 5}U/ml.

Figure 1 1 shows the lack of toxicity of recombinant OvIFN-tau on cultures of human peripheral blood mononuclear cells (PBMC), compared to interferon-alpha. PBMC were plated in 6-well plates at a concentration of 5 x 10⁶ cells/ml in a final volume of 2 ml. IFNs were added at the concentrations indicated and cells were counted and assessed for viability at 96 hours b y trypan blue dye exclusion. Human IFN-alpha was tested alongside the IFN-tau preparations.

DETAILED DESCRIPTION OF THE INVENTION

The starting point for the construction of the artificial gene is the amino sequence of OvIFN-tau identified by Imakaw a et al. (1987). The amino acid sequence includes 172 amino acids. In the current invention an artificial gene was designed to optimize OvIFN-tau expression in yeast, in particular Pichia pastoris. The strategy in this design included: (i) reducing repetitive sequences; (ii) lowering overall G+C content; (iii) reducing or eliminating palindromic sequences; and (iv) optimizing the nucleotide sequence using the biased codon usage of Pichia pastoris.

The codon usage of Pichia pastoris is well known to those skilled in the art. Since the nucleotide sequence presented herein could further be optimized to express the protein in various yeast species that have close or similar biased codon usage, the methodology of the present invention can be applied for expressing the same protein in all yeast species in general such as Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Yarrowia sp., Pichia sp., and Candida sp. One of ordinary skill in the art would know and use sequence analysis packages such as those from the Sanger Centre. These include applications such as palindrome, equicktandem, etandem, and geecee. According to these criteria artificial oligonucleotides were designed (Table 1) and an artificial OvIFN-tau gene w as constructed by polymerase chain reaction (PCR).

One of skills in the art would also know and us e plasmids, such as pPICZαA and pPIC9K (Invitrogen, CA) containing the AOX1 inducible promoter and the powerful α-MF prepro signal sequence. These expression vectors are replicable in E. coli and are used widely for gene expression in Pichia pastoris (Cereghino et al., 2000). A skilled artisan will readily understand how to engineer and to prepare variants of the sequence in th e present invention using methods known in the art such as, for example, by PCR or mutation (e.g. substitution, deletion, addition, insertion) without changing the properties thereof, an d particularly to prepare nucleotide sequences which code for the same amino acid sequence owing to the degeneracy of the genetic code (Sambrook et al., 1989).

As used herein the term "OvIFN-tau" refers generally to any OvIFN-tau amino acid or nucleic acid sequence including, for example, known sequences and those variants whose genes are characterized by a high degree of homology with the known original sequence of OvIFN-tau (Imakawa et al., 1987) and which code for biologically active OvIFN-tau and compounds having substantially the same biologically activity as known forms of OvIFN-tau. A whole series of 11 IFN-tau sequences from various species (66 to 95% amino acid sequence homology to rOvIFN-tau) which code for members of the IFN-tau family are known

(Stewart et al., 1989; Klemann et al., 1990; Nephrew et al., 1993 ; Leaman et al., 1992).

In the present invention, a nucleotide sequence (SEQ ID NoJ3) which codes for rOvIFN-tau having an amino acid sequence of SEQ ID NoJ6 shown in Figure 1A was disclosed. The SEQ ID NO. 13 has the restriction map of SEQ ID No. 14. Preferably, the ovine interferon-tau in the methods and constructs of the present invention is encoded by the nucleotide sequence of SEQ ID No. 15. It is further preferred that in the methods and constructs of the present invention the ovine interferon-tau is encoded by a nucleotide sequence that is at least about 70% homologous with the nucleotide acid of SEQ ID No. 15 , particularly a nucleotide sequence which codes for a protein having recombinant ovine interferon-tau activity.

It is also preferred to use the pPICZαA or pPIC9K expression vectors from Invitrogen which had not been u sed before for the production of rOvIFN-tau. Detailed features of these vectors are available from the manufacturer. It is in particular preferred in the present invention that Pichia pastoris alcohol oxidase I promoter (AOXl) is used for controlling expression. The isolation of the alcohol oxidase and the sequence of the AOXl promoter were disclosed by Ellis et al. (1985) and Koutz et al. (1989). In order to secrete the protein it is preferred to use the secretion signal sequence from the Saccharomyces cerevisiae α factor prepro peptide (α-MF). It is also preferable to include a stop codon signal downstream of the gene in order to get the expected native C-terminal amino acid sequence. One of skill in the art can easily produce variants from the sequences in the expression vector, particularly the promoter and the secretion signal sequence α-MF.

It should be noted that when using the pPICZαA or pPIC9K vectors of the present invention, the N- terminal processing of the α-MF signal peptide is not accurately processed. Consequently, in vivo N-terminal processing of the recombinant protein encoded by constructs pPICZαA-OvIFN-tau5 or pPIC9K- OvIFN-tau7 of the present invention may engineer two extra amino acids, namely a glutamic acid (position 1) and a phenylalanine (position 2). If needed, these two extra residues can be removed by either (i) using two sequential Edman reactions, (ii) modifying by mutagenesis the expression vector pPICZαA-OvIFN-tau5 or pPIC9K-OvIFN-tau7, or (iii) using another signal peptide such as PHOl (van Heeke et al., 1996) or PHA-E from the plant lectin Phaseolus vulgaris agglutinin (Raemaekers e t al., 1999) that has been recently shown to be efficient. However, these signal pep tides are not as widely used as the α-MF and generally give lower secretion of proteins (Cereghino et al., 2000). It should also be noted that these two additional residues at the N- terminus of OvIFN-tau have no effect on the antiviral activity of the protein. Construction of the synthetic sequence SEQ LD No.15, the cloning vector, and transformation of suitable E. coli strains can be carried out using methods known in the art (Sambrook e t al., 1989). For example, PCR method using overlapping oligonucleotides, as used by Casimiro et al. can be used to engineer the synthetic SEQ ID NoJ5. The E.coli strain TOP10F' (E. coli F {lacF, Tn70(Tet^R)} rcA A(mrr-hsdRMS-mcrBC)<$, 80 /αcZΔM15 AlacX74 deoR recAl araO139 A(ara-leu)7697 galU galK rpsL(Stι^R) endAl nupG) is particularly suitable for cloning gene. However, a skilled artisan may reasonably utilize other available E.coli strains including, for example, DH5α or JM109.

It is preferred for Pichia pastoris protein expression to use the Invitrogen recommendation (EasySelect™ Pichia Expression Kit, Version A, Invitrogen). For transformation and protein expression experiments, the X33 wild-type (Mut⁺, His⁺) strain or GS115 (His⁺, Mut^") strain can be used. The X33 strain was not used before by others for rOvIFN-tau expression. Expression can reliably be controlled by, for example, monitoring the dissolved oxygen (DO) in the medium (DO>30%). In bulk flask experiments, only methanol needs to be supplied during th e induction phase. It was observed that concentration of methanol needs to be relatively low during the methanol adaptation phase (0.4%, 4h) and slowly increased by increment of 0.2% up to 1.5% until 4 days of induction. After 4 days, the methanol concentration needs to be decreased for the next 24 hours. The recombinant protein is secreted in large amounts into th e periplasmic space. Based on 1D-NMR experiments from purified bulk flask and fermentation samples the secreted protein is correctly folded and has correct disulfide bridges. The 1D-NMR spectra results are fully in accordance with prior 1D-NMR spectrum recorded for protein expressed by GS 115/#29.

Nucleotide sequencing experiments showed that the protein expressed by both vectors of the present invention has two extra amino acids, namely a glutamic acid and a phenylalanine, at the N-terminal of the protein. From PAGE- Tricine gel electrophoresis, MALDI-TOF MS, CD spectra and 1D- NMR analysis, rOvIFN-tau expression in X33 or GS115 showed a 70-80% purity from the medium and over 95% after a double step purification.

It is preferred that one of skill in the art compares th e expression of rOvIFN-tau using the transformant presented herein with the transformant GS 115/#29 from van Heeke et al. ( 1996) . This transformant GS 115/#29 was transformed with a multicopy pMLD3031Stl l vector containing the PHOl (Pichia pastoris acid phosphatase) signal sequence and the original OvIFN-tau amino acid sequence described by Imakawa et al. (1987).

The initial extraction methods useful in the present invention are known in the art (see, for example, Sambrook et al., 1989 and those described by van Heeke et al. 1996). However, an extraction method which is preferred in the invention involves a double-step saturation with ammonium sulfate (20% and 80%) a t 4°C. The final pellet is suspended in Tris-HCl 20 mM, pH 7.6 and dialyzed against a Tris-HCl 1 mM, pH 7.5 solution for 2 days, filtered using Microcon-3kDa cut-off (Amicon) and concentrated 10-fold before addition to a Q-Sepharose Fast Flow column (Pharmacia) equilibrated with a Tris-HCl 20 mM, pH 7.6 solution. It is preferred that the flow rate of the column be 3 ml/min. A buffer, especially concentrations ranging from 150 to 350 mM NaCl is preferred as an eluant. Purified fractions analyzed by SDS- Tricine 15% are pooled, dialyzed against 25 mM Tris-HCl, ρH7.6, filtrated and concentrated using a 3 kDa Amicon membranes.

It is preferred that purified proteins are run through a gel filtration column. Type HR100 Sephacryl gel made by Sigma is preferred. It is also preferred that the column is equilibrated with Tris-HCl 20 mM buffer, pH7.6. The rOvIFN-tau is preferably eluted at pH7.6 using 20 mM Tris-HCl, and a linear gradient of 0 - 250 mM NaCl.

It is suggested that the skilled artisan pools the purified fractions together and filtrate the purified proteins through a 3-kDa Amicon membrane. The purified proteins can b e then dialyzed against 10 mM Tris-HCl, pH7.6 for 48 hours, against 5 mM Tris-HCl, pH7.6 for 24 hours, 2.5 mM Tris-HCl, pH7.6 for 24 hours, and finally against dH₂0 for 6 hours. For long term storage the purified rOvIFN-tau can be lyophilized or suspended at a concentration from 2 to 5 mM in a solution made of 10 mM Tris- HCl pH7.6, 2% ammonium sulfate, and 0.2% sodium azide. All purified protein samples should be stored at -20°C.

Further analysis of the purified rOvIFN-tau samples can be performed by gel electrophoresis, especially an SDS-Tricine polyacrylamide gel under standard conditions, particularly a 15% gel. The rOvIFN-tau content of various samples obtained during purification may be quantitated by any technique known in the art for protein quantitation, particularly the Bradford and th e Lowry methods (Sambrook et al. 1989).

Other characterization of the rOvIFN-tau can b e evaluated further. A skilled artisan can refer to MALDI-TOF Mass spectrometry experiment to assess the level of purity and the correct molecular weight of the protein. In order to evaluate the correct folding of the recombinant ovine interferon-tau as expressed herein in Pichia pastoris, a skilled artisan can use one dimensional Nuclear Magnetic Resonance (1D-NMR) and compare the spectrum to the one performed using the same protein expressed by GS 115/#29. To assess the secondary structure of the protein, circular dichroism (CD) spectrum of 15N-labeled OvIFN-tau can be recorded.

To assess the biological activity of rOvIFN-tau, ordinarily a skilled artisan can perform antiviral assay using th e cytopathic effect protection and toxicity procedures. The antiviral assay consists of using animal cell lines such as Madi-Darby Bovine Kidney (MDBK) cells challenged with virus such a s stomatitis virus as described by Pontzer et al. (1988). The toxicity assay can be performed on cultures of human peripheral blood mononuclear cells (PBMC). PBMC can be plated at a concentration of 5 x 10⁶ cells/ml. Recombinant OvIFN-tau was added a t different concentrations (1 to 5 x 10⁷ U/ml) and cells w ere counted and assessed for viability at 96 h by trypan blue dy e exclusion. Human interferon-alpha can be used as a control. In one embodiment of the present invention, there is provided an artificial ovine interferon-tau gene designed for high yield protein production in yeast, wherein the gene is constructed by (i) reducing repetitive sequences; (ii) lowering overall GfC content; (iii) reducing or eliminating palindromic sequences; and (iv) optimizing the nucleotide sequence of the gene using th e biased codon usage in yeast. In general, the methodology is applicable as is for Pichia sp. , or by appropriate optimization such as optimizing the specific biased codon usage to other yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Yarrowia sp. and Candida sp. More preferably, the recombinant gene has the sequence shown in SEQ ID No. 13 or 15.

In another embodiment of the present invention, there is provided two expression vectors designed for high yield ovine interferon-tau production in Pichia pastoris, said vectors comprising (a) an artificial ovine interferon-tau gene constructed by (i) reducing repetitive sequences; (ii) lowering overall GfC content; (iii) reducing or eliminating palindromic sequences; and (iv) optimizing the nucleotide sequence of the gene using the biased codon usage in yeast; (b) a Saccharomyces cerevisiae α- mating polypeptide signal sequence for efficient secretion of protein into the media; (c) an inducible alcohol oxidase I promoter; and (d) regulatory sequences effective for expressing said interferon-tau in said yeast. More preferably, the expression vector is pPICZαA-OvIFN-tau or ρPIC9K-OvIFN-tau. In yet another embodiment of the present invention, there is provided a method for high yield production of ovine interferon-tau in Pichia pastoris, comprising the steps of: transforming said yeast with the expression vectors disclosed herein; inducing protein expression with methanol; culturing said yeast in defined culture conditions; and purifying the protein from the culture media. Preferably, the yeast is Pichia pastoris X33 or Pichia pastoris GS115. The yeast is cultured in shake flask or fermentor. Having now generally described this invention th e same will better be understood by reference to certain specific examples which are included for the purposes of illustration an d are not intended to limit the invention unless otherwise specified.

EXAMPLE 1

Preparation of the Synthetic OvIFN-tau Gene Nucleic acid amplification (PCR), restriction digestion of

DNA with restriction endonucleases, phenol extraction and precipitation of DNA, agarose gel electrophoresis and purification of DNA from agarose gels, ligation of DNA molecules, transformation of bacteria and plasmid isolation from bacteria are standard procedures and were carried out as described b y Sambrook et al. (1989). EXAMPLE 2

Expression Vectors

Expression vectors pPICZαA-OvIFN-tau and pPIC9K- OvIFN-tau were prepared from the expression plasmids pPICZαA and pPIC9K respectively (Invitrogen). Each contains the promotor of the alcohol oxidase namely AOXl from Pichia pastoris (Ellis e t al., 1985), the coding region of the α-mating factor secretion peptide (α-MF) from Saccharomyces cerevisiae (Singh et al., 1983) and the new gene encoding for ovine IFN-tau. Figures 2A and 2B show the pPICZαA-OvIFN-tau and pPIC9K-OvIFN-tau expression vector maps.

EXAMPLE 3

OvIFN-tau Gene Engineering

A new gene encoding for OvIFN-tau was engineered using polymerase chain reaction (PCR). Overlapping megaprimers were designed according to the following guidelines: (i) reducing or eliminating palindromic sequences that would lead to stable intramolecular hairpins; (ii) lower overall G+C content (<50%) to improve transcriptionnal level event; (iii) minimum sequence repeats when possible, and (iv) biased use of Pichia pastoris codons. In order not to compromise the first 3 design requirements, the two most used Pichia codons were utilized for each amino acid (except Met and Trp), instead of just the best one. This also allows access to a larger tRNA pools during protein synthesis. EXAMPLE 4

Megaprimers and Preparation of the OvIFN-tau Sequence in a Two-Step PCR Reaction

Table 1 depicts the megaprimers used in PCR reaction employed in the construction of the new synthetic OvIFN-tau gene. Megaprimers were synthetized by Integrated DNA Technologies Inc. (Coralville, IA) on a 0J μmol scale and purified by PAGE. Prlf to PrlOr (SEQ ID No.l to SEQ ID No.lO, Table 1 ) were used for the first PCR reaction (PCRla). The PCR reaction was carried out on a Progen DNA Thermal Cycler (Progen). The first PCR reaction contained 10 pmol each of the ten oligonucleotides, 2.5 units of Deep Vent polymerase (Biolabs Inc.) and 0.25 mM of each deoxynucleotide triphosphate (dNTP) in 1 00 μl volume. The solution was buffered in 60 mM Tris-HCl, 15 mM (NH₄)₂S0₄, and 2.5 mM MgCl₂, pH 8.5 (22°C). The reaction involved a hot start at 85°C for 1 min followed by 30 cycles of 30 s at 94°C, 30 s at 64°C, and 1 min 20 s at 72°C. The reaction was completed with a 5 min incubation at 72°C. Ten microliters of this reaction mixture was then transferred to a second PCR tube containing two oligonucleotides PrAf (26-bases) and PrBr (25-bases) (100 pmol each) representing the outermost 5' sequences of both strands (Table 1).

The second PCR, namely PCRlb, was buffered as described above. The mixture was incubated at 94°C prior to adding 2.5 units of Deep Vent polymerase (Biolabs Inc.) and 0.25 mM of each deoxynucleotide triphosphate (dNTP). The reaction was followed by 30 cycles of 30 s at 94°C, 50 s at 55°C, and 1 min 20 s at 72°C. Reaction was further completed with a 5 min incubation at 72°C. The resulting DNA fragment of PCRlb (5 19 bp) was gel-purified in 1.5% low-melting agarose in TAEIX buffer (TAEIOX: Tris 48.4 g, Na₂EDTA (2 H₂0), glacial acetic acid 11.4 ml). The agarose section containing the DNA fragment of PCRlb w as excised and the agarose was melted at 50°C for 5 min. The DNA mixture was further purified using the QIAEX II purification kit (Qiagen Inc, Valencia, CA).

TABLE 1

Primers used to engineer the rOvIFN-tau gene SEOIDNO:! (Prlfi 5^NTCG TAC GAG CTC AGA CAG CTG GGT ACC GCG GAA TTC TGT TAG TTG TCT CGT AAG TTG ATG TTG GAC GCT CGT GAA AAC TTG A 3

SEOIDNO:2 CPτ2τ)

5 TTA CGA TCC TGC AAA CAG GAG TGT GGA GAC AAA CGG TTC ATA CGA TCC AAC AAT TTC AAG TTT TCA CGA GCG TCC AAC ATC AA 3

SEOIDNO:3 (Pr3fJ

5 CAC ACT CCT GTT TGC AGG ATC GTA AGG ATT TCG GTT TGC CTC AAG AAA TGG TTG AGG GTG ATC AAT TGC AGA AGG ACC AAG CTT 3

SEOIDNO:4 (Pr4r)

5 ATG TTC AGT GTA GAA CAA GTT AAA AGA CTG TTG CAA CAT TTC

ATA CAA GAC TGG AAA AGC TTG GTC CTT CTG CAA TTG ATC A 3

SEOIDNQ.5 (Pr5fJ

₅. TCT _{ττχ AAC TTG τχc TAC Acτ GAA CAT τcτ τcτ GCC GCT TGG GAχ}

ACT ACA TTG CTT GAA CAA TTG TGC ACT GGA CTT CAA 3

SEOTDNO:6 (Pr6r 1 5^{^} TCA GAA TCC TCT TCA CCC ATA CCT TGT CCA CGA CAA GTA TCC AAA TGA TCC AAT TGC TGT TGA AGT CCA GTG CAC AAT TGT 3

SEOTDNO:7 (Pr71J

5 AAG GTA TGG GTG AAG AGG ATT CTG AAT TGG GTA ACA TGG ATC CTA TTG TCA CTG TTA AGA AAT ACT TTC AAG GTA TCT ACG ACT 3^N

SEOTDNO:8 (Pr8r^

5^V ATC TCA ACA CGG ACA ATT TCC CAA GCA CAA TCA GAA TAA CCT

TTC TCT TGA AGG TAG TCG TAG ATA CCT TGA AAG TAT TTC T 3^{^} SEQ ID NO:9 (Pr9tJ

5^{^}CTT GGG AAA TTG TCC GTG TTG AGA TGA TGC GTG CCT TGA CTG TTT

CCA CAA CTC TTC AAA AGC GTT TGA CTA AGA TGG GAG GTG A 3

SEOIDNO:10 (Pr IQi

5 TCA TCT GGT CAG CGG CCG CAT TGG AGT CTA GAT TAG TTA TGG AGA GTT CAA ATC ACC TCC CAT CTT AGT CAA ACG CTT TT 3 ^s

SEOIDNO:!! (PrA (f)) 5 TCg TACgAg CTCAgA CAg CTg ggTAC3

SEOIDNO:12 (PrB (r))

5 TCA TCT ggT CAg Cgg CCg CAT Tgg A 3^{^}

EXAMPLE 5

Preparation of the Expression Vectors pPICZαA-OvIFN-tau and pPIC9K-OvIFN-tau

For the construction of the expression vectors pPICZαA-OvIFN-tau and pPIC9K-OvIFN-tau, plasmids pPICZαA, pPIC9K, as well as the PCR product (OvIFN-tau) were respectively doubly cut with EcoRI and Notl. After digestion, 50 ng of purified plasmid and 150 ng of PCR product were ligated in a 10 μl volume containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 20 mM dithiothreitol, 1 mM ATP, 50 mg/ml bovine serum albumin (BSA) and 2 units of T4-DNA-ligase (Biolabs Inc.) for 6 hour at 4°C and 1 hour at ambient temperature. Competent cells TOP10F' (F' {lacl^q Tnl0(Tet^R)} mcrA Δ(mrr-hsdRMS-mcrBC) φ801acZΔM15 ΔlacX74 deoR recAl araD139 Δ(ara-leu)7697 galU galK rpsL (Str^R) endAl nupG) were transformed with 5 μl of the ligation reaction. Plasmid DNA from various colonies thus obtained was isolated and checked for correct composition by restriction analysis. A clone for each construction was selected, the DNA was isolated and the expression cassette sequenced. The constructions from clones # 5 and #7 were designated pPICZαA-OvIFN-tau5 pPIC9K-OvIFN-tau7 respectively. Both constructs were checked by DNA sequencing.

EXAMPLE 6

Transformation in Pichia pastoris X33 and GS115

The expression vector pPICZαA-OvIFN-tau5 w as linerized in 5' of the AOXl with Pmel. The expression vector pPIC9K-OvIFN-tau7 was linerized in 5' of the AOXl with Bglll. About 15 μg of each expression vector was digested and further purified by phenol/chloroform extraction and ethanol precipitation. Pichia pastoris X33 and GS115 strains w ere cultured in order to prepare competent cells as recommended b y the manufacturer instructions (Invitrogen). The DNA w as suspended in 5 μl of dH₂0 and transformed by electroporation into 50 μl of Pichia pastoris X33 (Mut⁺, His⁺) or GS115 (His⁺, Muf) suspended in cold 1 M sorbitol. After electroporation, cells w ere immediately suspended into 1 ml of ice-cold 1 M sorbitol, transferred in a 14-ml polypropylene fresh tube and incubated a t 30°C for 2 hours without agitation. Cells were therefore plated in yeast peptone dextrose agar-plates containing Zeocin (100 μg/ml for X33 transformants and 0.25 to 1 mg/ml for GS 115 transformants) and incubated at 30°C for 2-3 days. Numerous Pichia pastoris transformants were selected and screened for rOvIFN-tau protein expression.

EXAMPLE 7

rOvIFN-tau Expression in Shake Flasks

According to the Invitrogen Inc. recommendations, transformants were first screened in a small-scale experiment ( 5 ml) using BMGY and later using BMMY media for protein induction and expression. Transformant X33/OvIFN-tau3 demonstrated th e highest expression in the medium after 4 days of 0.4 to 1.5% methanol induction and was utilized for scale-up experiments . From a 24 hours initial culture (50-ml), transformants X33/pPICZαA-OvIFN-tau3, GS 115/pPIC9K-OvIFN-tau4, as well a s the van Heeke et al. transformant GS115/#29 were grown at 30°C, 300 rpm, for 24 hours in a 1-liter scale in BMGY media containing 20 g of peptone, 10 g of yeast extract, 50 ml of 1M potassium phosphate buffer, pH 6, 10 mg biotin, and 2% glycerol. The culture was briefly spun down at 5,000 rpm and suspended into the BMMY induction medium that has the same composition a s BMGY except for replacing 2% glycerol by 0.5% methanol. From the induction starting point methanol (0.2 to 0.5%) w as supplemented after 18, 36, 48, 60, 72 and 96 h. rOvIFN-tau expression from transformants X33/pPICZαA-OvIFN-tau3, GS 115/pPIC9K-OvIFN-tau4, or GS 115/#29 were screened through the induction period in performing a 15% PAGE-Tricine. EXAMPLE 8

rOvIFN-tau Expression in a 28-L Fermentor Fermentation experiments were carried out using a

MICROFERM^® fermentor 28L (New Brunswick, NJ, USA). The fermentor was initially supplemented with 16-L of media comprising 2.27% H₃P0₄ (85%), 6.83 mM CaS0₄, 105 mM K₂S0₄, 5 8 mM MgS0₄ (7H₂0), 73.6 mM KOH, 3% Glycerol, 10 mg/1 Biotin, and PTMl salts 4ml/l. The pH was adjusted to 5.5 with a 30% NH₄OH solution. After autoclaving the fermentor 40 min at 120°C and cooling down to 30°C, a 500-ml rOvIFN-tau culture was inoculated and cultured overnight. Temperature was set up at 29.5°C ± 0.5 and pH to 5.5 ± 0.2 using a 30% NH₄OH solution. The dissolved oxygen (DO) was kept above 30% varying the agitation, and the pressure maintained at 7 psi. The next day glycerol (30%) was fed from 1 to 2.5 ml/min for 12 hours. Supplementation of pure 0₂ was carried out to maintain DO between 30-120%. After 12 hours, supplementation of glycerol was stopped. Depletion of glycerol was effective when a transient DO spike was observed. Methanol feeding was then started at 0.5 to 2 ml/min for 4-5 days. At the end of the fermentation, the cells were centrifuged at 4,000 rp m at 4°C for 15minutes. For medium term storage at 4°C (typically 1-3 months), 20% ammonium sulfate and 0.2% sodium azide can be added to the supernatant until further experiments. EXAMPLE 9

OvIFN-tau Purification

Anion-Exchange Chromatography Purification Process Prior to purification, the protein suspension can be extensively washed and concentrated by filtration through a 3-kDa Amicon membrane with a 20 mM Tris-HCl, pH 7.6 solution. Proteins were loaded on a 800 mL Q-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) column equilibrated with 20 mM Tris-HCl, pH 7.6. Elution was carried out using 20 mM Tris-HCl, NaCl 0.35 M pH 7.6 at a flow rate of 3 ml/min.

Gel Filtration Purification Process The Q-

Sepharose, material was dialyzed against 25 mM Tris-HCl, pH7.6, filtrated and concentrated by 3-kDa Amicon membrane to 30 to 40 mg/ml. The protein material was then purified over a 1.5 x 120 cm Sephacryl S-100 HR column equilibrated in 20 mM Tris- HCl, pH 7.6. Proteins were eluted at a flow rate of 1 ml/min using 20 mM Tris-HCl, NaCl 0.25 M, pH7.6. Fractions showing the purified fraction on 15% PAGE-Tricine were pooled, dialyzed against 10 mM Tris-HCl, pH 7.6 for 48 hours, against 5 mM Tris- HCl, pH 7.6 for 24 hours, against 2.5 mM Tris-HCl, pH 7.6 for 24 hours, and finally briefly (6 hours) against dH20. The concentrated proteins can be lyophilized or suspended in a solution made of 5 or 10 mM Tris-HCl, pH 7.6, 2-5% ammonium sulfate, and 0.2% sodium azide. Proteins can be kept at -20°C at a concentration of 1 to 5 mM until further experiments. EXAMPLE 10

Analysis of the rOvIFN-tau Preparation Biological Activity Antiviral activity of rOvIFN-tau was determined using a cytopathic effect protection assay using Madi-Darby Bovine Kidney (MDBK) cells challenged with vesicular stomatitis virus, as described by Pontzer et al. (1988). One unit of antiviral activity was defined as the reciprocal of the dilution of IFN sufficient to reduce the cytopathic effect of VSV by 50%. The toxicity assay was performed on cultures of human PBMC. PBMC were plated in 6-well plates at a concentration of 5 x 10⁶ cells/ml in a final volume of 2 ml. Recombinant OvIFN-tau was added at the concentrations indicated and cells were counted and assessed for viability at 96 hours by trypan blue dye exclusion. Human IFN-alpha was tested alongside the OvIFN-tau preparations.

MALDI-TOF Mass Spectrometry Correct mass (about 20.4 kDa) of the purified rOvIFN-tau was confirmed by Mass Spectrometry MALDI-TOF analysis (PerSeptive Biosystems). The acceleration voltage was set at 25 kV. Samples were mixed 1 / 1 0 with sinapininc acid dissolved in acetonitrile: 0.1% trifluoroacetic acid (1 :1). Apomyoglobulin, thioredoxin, and insulin were used to standardize the mass spectrometer.

One Dimensional Nuclear Magnetic Resonance (ID- NMR) The globular protein conformation of this protein in solution was confirmed by 1D-NMR experiments performed at 25°C on a 500 MHz or a 600 MHz Bruker NMR instrument. Lyophilized protein was resuspended in 5 mM deuterated Tris- HCl, pH 7.5 with 10% D₂0 to a concentration of 1 mM. ID-NMR spectra of correct folded rOvIFN-tau was characterized by well- resolved ring-current shifted resonances in the methyl group region and the aromatic proton region. The ID-NMR spectra for the preparations of the present invention were identical to the control ID-NMR spectrum obtained on a biologically active rOvIFN-tau from GS115/#29 (data not shown).

Circular Dichroism Circular dichroism spectrum of

¹⁵N-labeled OvIFN-tau at 1.5 mg/ml, pH 7.2, Tris-HCl 1 mM, w as recorded at 20 C on an Aviv 62DS spectropolarimeter using 0.01 mm cell. Five scans from 260 to 185 nm were averaged, using a 2-mm bandwidth, 1 second averaging time, and 0.5 mm intervals.

Polyacrylamide-Tricine Gel Electrophoresis rOvIFN- tau samples were analyzed on 15% polyacrylamide-Tricine gel electrophoresis under standard reducing conditions as previously described (Schagger et al., 1987). Proteins bands were visualized with Coomassie blue staining.

EXAMPLE 11

Results

Using the engineered synthetic OvIFN-tau nucleotide sequence, the expression vector pPICZα-IFN-tau3, and the expression process described in Example 3, it was possible to obtain as high as 690 mg/1 and 1.35 g/1 of purified rOvIFN-tau in shake flask and after fermentation experiments respectively. Flask experiments using pPIC9-IFN-tau7 demonstrated a lower expression (584 mg/1 in shake flask). Expression results in th e present invention were compared with the expression of th e transformant GS 115/#29 from van Heeke et al. Expression in bulk flasks for GS 115/#29 demonstrated a yield of only 66 m g/1 of culture. Thus, the present expression results in bulk flask using X33/pPICZαA-OvIFN-tau3 represent at least 10 times production improvement.

Fermentation experiment results using X33/pPICZαA- OvIFN-tau3 enhanced protein production by 5 times in comparison with GS115/#29 (1.35 g/1 vs 0.28 g/1 respectively). I t should be noted that the fermentation experiment results disclosed herein could be significantly improved further using a fermentor that allows higher agitation (up to 700 rpm) and higher cell density, thereby getting even more rOvIFN-tau expression in the medium. In order to get even higher cell density, a skilled artisan can also increase the glycerol Fed-Batch step up to 3 6 hours .

The fact that there was also significant improvement of rOvIFN-tau production using GS 115/ρPIC9K-OvIFN-tau4 (9x in shake flask) indicates that the vector construction and the ne w OvIFN-tau gene itself have a strong effect on the rOvIFN-tau expression. As observed by PAGE-Tricine and MS MALDI-TOF spectrum, Q-Sepharose and HR100 Sephacryl chromatography led to a r OvIFN-tau pool with a purity over 95%. Figures 5 A and 5B show characteristic chromato grams of each purification step and Figure 5C shows the level of purity (>95%) after the double purification step.

The rOvIFN-tau expressed by transformants

X33/pPICZαA-OvIFN-tau3 and GS115/pPIC9K-OvIFN-tau4 was further characterized. PAGE-Tricine and MS MALDI-TOF showed the correct size for the protein (approximately 20.4 to 20.5 kDa). Correct folding of the protein expressed in shake flasks or fermentation was demonstrated by ID-NMR and illustrated in Figures 8A and 8B. A CD spectrum was also recorded (Figure 9). It confirms the presence of a protein with a stable secondary structure dominated by β-helices.

The purified rOvIFN-tau from flasks or fermentation experiments demonstrated full anti-viral activities against vesicular stomatitis virus (VSV) on Madi-Darby Bovine Kidney (MDBK) cells using a cytopathic effect microplate assay. Activities of 1 x 10⁸ and 1 x 10^{7 5} antiviral units/ml were detected in the purified samples from shake flasks and fermentation experiments respectively. Figures 10A and 10B show the anti-proliferative assay results of the purified rOvIFN-tau from shake flask and fermentation experiments. Same biological activity results were obtained using either X33/pPICZαA-OvIFN-tau3 or GS 115/pPIC9K-OvIFN-tau4. To assess the level of toxicity of the rOvIFN-tau from bulk flask and fermentation experiments, a toxicity assay (Figure 11) was performed on cultures of human peripheral blood mononuclear cells (PBMC). Results demonstrated that PBMC cells have greater than 80% of viability when using a concentration range of 1 to 10⁷ U/ml. This lack of toxicity was however not observed at high concentrations when using IFN- alpha. Stability of the lyophilized OvIFN-tau or after long-term storage in solution at -20° C was demonstrated by repeatitive 1D- NMR analysis, antiviral and toxicity assays.

The following references were cited herein:

Casimiro et al., Biochemistry. 34:6640-6648 (1995).

Cereghino et al., FEMS Microbiology Reviews. 24:45-66 (2000). Ellis et al., Mol. Cell. Biol. 5: 1111-1121 (1985).

Imakawa et al., Nature 330:337-379 (1987).

Johnson et al., J. Interferon Cyt. Res. 19:631-636 (1999).

Klemann et al., Nucl. Ac. Res. 18:6724-6726 (1990).

Koutz et al., Yeast 5:167-177 (1989). Leaman et al., J. Interf. Res. 12: 1-11 (1992).

Nephrew et al., Biol. Reprod. 48:768-778 (1993).

Ott et al., J. Interferon Cyt. Res. 11 :357-364 (1991).

Pontzer et al., Biochem. Biophys. Res. Commun. 152 : 801 - 807

( 1988). Raemaekers et al., Eur. J. Biochem. 65:394-403 (1999).

Sambrook et al., (1989) Molecular Cloning - A laboratory Manual,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Schagger et al., Anal. Biochem. 166, 368-397 (1987).

Singh et al., Nucleic Acids Res. 11 :4049-4063 (1983). Stewart et al., J. Mol. Endocrinol. 2:65-70 (1989). van Heeke et al., J. Interferon Cyt. Res. 16: 119-126 (1996).

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the s ame extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled in the art will appreciate readily that th e present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments , molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined b y the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. An artificial ovine interferon-tau gene designed for high yield protein production in yeast, wherein said gene is constructed by:

(i) reducing repetitive sequences in said gene; (ii) lowering overall G+C content in said gene; (iii) reducing or eliminating palindromic sequences in said gene; and (iv) optimizing the nucleotide sequence of said gene using the biased codon usage in said yeast.

2. The gene of claim 1, wherein said yeast is selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces sp., Yarrowia sp., Pichia sp., and Candida sp.

3. The gene of claim 2, wherein said Pichia sp. is

Pichia pastoris.

4. The gene of claim 1, wherein said gene comprises of sequence selected from the group consisting of SEQ ID No. 1 3 and SEQ ID No. 15.

5. An expression vector designed for high yield ovine interferon-tau production in Pichia pastoris, said vector comprising: an artificial ovine interferon-tau gene constructed b y (i) reducing repetitive sequences in said gene; (ii) lowering overall G+C content in said gene; (iii) reducing or eliminating palindromic sequences in said gene; and (iv) optimizing the nucleotide sequence of said gene using the biased codon usage in Pichia pastoris; a Saccharomyces cerevisiae α-mating polypeptide signal sequence for efficient secretion of protein; an inducible alcohol oxidase I promoter; and regulatory sequences effective for expressing said interferon-tau in Pichia pastoris.

6. The expression vector of claim 5, wherein said artificial ovine interferon-tau gene comprises of SEQ ID No. 15.

7. The expression vector of claim 5, wherein said vector is selected from the group consisting of pPICZαA-OvIFN-tau and pPIC9K-OvIFN-tau.

8. A method for high yield production of ovine interferon-tau in the yeast of Pichia sp. , comprising the steps of: transforming said yeast with the expression vector of claim 5; inducing protein expression with methanol; culturing said yeast in defined culture conditions; and purifying said protein from the culture media.

9. The method of claim 8, wherein said yeast of Pichia sp. is selected from the group consisting of Pichia pastoris X33 and Pichia pastoris GS 115.

10. The method of claim 8, wherein said culture conditions for culturing said yeast is selected from the group consisting of shake flask and fermentor.