WO2001077351A1

WO2001077351A1 - Vectors and methods for dual protein expression in pichia pastoris and escherichia coli

Info

Publication number: WO2001077351A1
Application number: PCT/EP2001/003995
Authority: WO
Inventors: Angelika LÜKING; Caterina Holz; Hans Lehrach; Dolores Cahill
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2000-04-07
Filing date: 2001-04-06
Publication date: 2001-10-18

Abstract

The present invention relates to a shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising: a promoter which is a yeast alcohol oxidase (AOX) promoter, a yeast CUS1 promoter, (ac) a tetracycline promoter; or (ad) a CMV promoter, an E. coli T7 promoter; a Pichia pastoris autonomously replicating sequence (PARS); and a multiple cloning site. The present invention also relates to a host cell transformed/transfected with the shuttle vector of the present invention, to a collection of host cells comprising a collection of shuttle vectors of the invention, and to a method of producing a (poly)peptide comprising culturing the host cell of the present invention under suitable conditions and isolating said (poly)peptide from the culture. Finally, the present invention relates to a method of rearraying clones comprising picking the collection of host cells of the present invention; and transferring said collection of host cells in arrayed form to a liquid medium or a solid support, to an array of clones that is obtainable by the method of the present invention, and to a kit comprising the vector; the collection of shuttle vectors; the host cell; the collection of host cells, and/or the array of the present invention, in one or more containers.

Description

Vectors and Methods for dual protein expression in Pichia pastoris and Escherichia coli

The present invention relates to a shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising: a promoter which is a yeast alcohol oxidase (AOX) promoter, a CMV promoter, a tetracycline promoter, or a CMV promoter, an E. coli T7 promoter, a Pichia pastoris autonomously replicating sequence (PARS), and a multiple cloning site. The present invention also relates to a host cell transformed/transfected with the shuttle vector of the present invention, to a collection of host cells comprising a collection of shuttle vectors of the invention, and to a method of producing a (poly)peptide comprising culturing the host cell of the present invention under suitable conditions and isolating said (poly)peptide from the culture. Finally, the present invention relates to a method of rearraying clones comprising picking the collection of host cells of the present invention; and transferring said collection of host cells in arrayed form to a liquid medium or a solid support, to an array of clones that is obtainable by the method of the present invention, and to a kit comprising the vector, the collection of shuttle vectors, the host cell, the collection of host cells, and/or the array of the present invention, in one or more containers.

The yeast Pichia pastoris and the bacterium E. coli are commonly used host systems for the expression of recombinant, heterologous proteins. The choice of the expression host determines the quality of the recombinant protein and is critical. Early successes in the production of heterologous proteins were achieved using the well-studied bacterium E. coll (Itakura, 1977). This prokaryotic expression system is simple to handle and reveals a cost-effective and high-level production of heterologous proteins. However, despite these advantages, expression of some genes often leads to the production of aggregated and denatured proteins, localized in inclusion bodies, with only a small fraction maturing into the desired native form (Marston, 1986; Makrides, 1996).

The methylotrophic yeast Pichia pastoris has been developed over the last few years into a powerful expression system for a number of foreign genes (reviewed in Cregg, 1993; Faber, 1995). Its ability for rapid growth at high cell density combined with the strong AOX promoter, has in some cases yielded up to several grams of the heterologous express protein per liter of culture (Cregg, 1993). This eukaryotic system has been used successfully for expression of soluble proteins (Cregg et al., 1993; Romanos, 1995).

However, these systems have their advantages and disadvantages, therefore choosing a suitable system has been a compromise depending primarily on the properties of the protein, the levels required, and its intended purpose. In the past, heterologous gene expression has often been treated empirically and a number of host organisms each requiring time-consuming and laborious subcloning into a number of expression vectors including different epitope tags of fusion proteins, which have to be subsequently tested for successful expression.

Therefore, the technical problem underlying the present invention was to provide methods and means that allow fast and efficient protein expression in both prokaryotic and eukaryotic systems at low costs.

The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising a promoter which is a yeast alcohol oxidase (AOX) promoter, a yeast CUS1 promoter, a tetracycline promoter, or a CMV promoter, an E. coli T7 promoter, a Pichia pastoris autonomously replicating sequence (PARS), and a multiple cloning site.

The term "Pichia pastoris autonomously replicating sequence" is described in Cregg, 1985 and allows the vector to be isolated from the cells, without having to be linearised. This increases the transformation efficiency from 1x10³ per μg DNA to 1x10⁵ per μg DNA. The vector of the present invention advantageously combines eukaryotic and prokaryotic promoter elements. Furthermore, by integration of a Pichia specific autonomous replicating sequence (PARS), vector linearization is no longer required. Thus small amounts of DNA are sufficient for transformation. Also, high transformation efficiencies up to 10⁵ clones per μg DNA (Cregg, 1985) can be obtained. This is especially useful for the transformation of for instance libraries or high throughput cloning/transformation. Moreover, the PARS sequence enables simple recovery of plasmids from yeast. Additional features include a Terminator Region (AOX-Ter) which is a region in the vector behind the inserted DNA.

The vector of the invention allows proteins to be expressed in high-throughput systems, without the necessity for large-scale sub-cloning. By choosing the appropriate expression system, e.g., soluble expressed proteins like growth factors or receptors can act as biotherapeutics and may be tested in clinical trials (Wittrup, 1999; Cregg, 1993; Romanos, 1992). It is also envisaged that such proteins will be a better target for the screening of antibodies that can then be used in diagnostic assays by recognizing structural epitopes. The soluble proteins are better sources for crystallization and NMR studies since they show probably a functional folding (Gaasterland, 1998). In addition, soluble expressed proteins are more suitable for the generation of native protein chips containing high density arrayed proteins. As could be shown in accordance with the present invention, 100% of the proteins, the cDNA of which has been subcloned into the vector of the invention, were successfully expressed by P. pastoris, whereas E. coli were able to express 86.2%. All proteins expressed in P. pastoris were soluble. Even proteins that were expected to be difficult to express, such as the highly aggregating -tubuline, the leucine- zipper containing FEZ1 or the mouse homologue zinkfinger protein 106 could be expressed in soluble form. Only 27.6% were soluble expressed in E. coli. However, E. coli produces high-levels of heterologous protein (1.5-20 μg/ml), whereas P. pastoris expresses less (0.5-5 μg/ml). Thus, the dual expression vector of the invention allows for the combination of all advantages of each system, and each system is immediately accessible without the need of time consuming sub-cloning procedures. E. coli produces large amounts of heterologous protein but it is well known to show certain problems like the accumulation and aggregation of proteins in inclusion bodies as a result of differences in the prokaryotic and eukaryotic codon usage. The use of the yeast expression system has the advantage of expression of soluble proteins.

As mentioned above, the use of proteins like growth factors, receptors, cytokines and hormones as drugs and biotherapeutics requires the soluble expression which has been shown to be successful in some cases in P. pastoris (Weiss, 1998; Glansbeek, 1998; Vollmer, 1996). A functional structure of the target protein is often necessary to study interactions with proteins, peptides, ligands, hormones, drug, an activator, an antibody, or chemical compounds, and to obtain structure-activity relationships by phage display technique. Within preclinical drug screenings those in vitro interaction studies can be used as a preselection of interacting chemical compounds linked with a reduction of cell-based assays. This may enable a high-throughput clinical screening (Dove, 1999 (Sept. 99); Fernandes, 1998; Rodrigues, 1997). Moreover, interesting proteins can be used to make monoclonal and polyclonal antibodies. The use of denatured purified proteins results in antibodies that may recognize only linear epitopes or motifs. The deficiencies in the bacterial host cell's ability to express soluble proteins and to perform post-translational modifications such as disulfide isomerization, phosphorylation or glycosylation diminish the applicability of bacterial protein expression as an expression system for proteins to be used for the generation and binding characterisation of antibodies or fragments thereof, or other protein binders. Since antibodies are widely used in diagnostic assays, the production of antibodies, and specifically monoclonal antibodies with high affinity and specificity to their antigen is desired. Furthermore, antibodies which recognize structural epitopes are viable drug lead candidates. Soluble expressed proteins are also a better source for crystallization and NMR structure analysis since non-water soluble proteins remained an unsolved problem (Gaasterland, 1998). Using robot technology, it is possible to make high density protein arrays on filters (Buessow et al., 1998) or chips (Lueking et al., 1999). Proteins expressed in E. coli can be purified in high-throughput under denatured conditions and can be spotted onto a protein binding surface (Lueking et al., 1999). In high-throughput, soluble proteins expressed in P. pastoris can be arrayed following purification under native conditions to generate a native protein chip, thereby facilitating chip-based binding and interaction (e.g. protein-protein interaction) assays. Combined with MALDI-TOF- MS, identification of binding ligands, drugs or compounds by mass spectrometry is envisaged (James et al., 1993; Pappin, 1993; Cottrell, 1994). It is also envisaged that the vector of the invention is used for the in vitro transcription/translation of cloned nucleic acid molecules, e.g., by using S30 lysate. Such methods are well known in the art (see, e.g., Sambrook et al.). In this embodiment, a termination signal such as, e.g., the E. coli T7 termination region may additionally be cloned downstream of the multiple cloning site into the vector of the invention to ensure efficient termination. This embodiment may thus be useful for the production of proteins that are toxic for the host cells used in accordance with the invention. The yeast CUS1 promoter is preferably from S. cerevisiae Koller et al., 2000. In accordance with the present invention, it was surprisingly found that the CUS1 promoter from S. cerevisiae leads to a significant reduction of the time of incubation when producing proteins in P. pastoris without, however, reducing the amount of protein produced as compared to the AOX promoter. In an example, when human GAPDH protein has been expressed using the CUP promoter 100 ng/ml of GAPDH protein was obtained (Koller et al., 2000).

In a preferred embodiment of the invention, the shuttle vector of the invention further comprises a nucleic acid sequence encoding a tag.

The term "tag" denotes, in accordance with the invention, a proteinaceous or peptidic sequence that can be detected either due to its capacity to emit a signal under suitable and generally established conditions or due to its capacity to react with a detectable further compound such as an antibody specifically recognizing the tag.

In a more preferred embodiment of the shuttle vector said tag is an oligo-histidine domain (His 6) ), GST-tag or a hybrid tag comprising his-tag and biotin tag. However, other tags like, e.g., c-myc, FLAG, alkaline phosphatase, EpiTag™, V5 tag, T7 tag, Xpress™ tag, hemaglutinin (HA)-tag, Strep-tag, or biotin, a fusion protein, preferably GST, cellulose binding domain, green fluorescent protein, yellow fluorescent protein, maltose binding protein or lacZ, can also be used in accordance with the invention. A further preferred tag is a hybrid tag of two tags, which are well-known in the art, namely a histidine tag and an in vivo biotinylation recognition site, which functions in both bacteria and yeast (Schatz, 1993; Smith et al., 1997).

Any of the above tag sequences and in particular the hybrid tag sequence may be followed by a trypsin recognition site which allows a unambiguous identification of proteolytic degradation products in mass spectrometric analysis.

A further preferred tag is GST-tag, which allows a very efficient purification from proteins produced in P. pastoris as shown in the figures and the appended examples.

In another more preferred embodiment of the shuttle vector said tag is a part of a fusion protein upon expression of said nucleic acid.

In this embodiment the tag may advantageously be used for the isolation and/or detection of the produced fusion protein.

In a most preferred embodiment said tag is positioned N-terminally of the fusion protein.

In another preferred embodiment of the shuttle vector of the present invention said E. coli T7 promoter is placed downstream from said yeast AOX promoter.

In a further preferred embodiment of the present invention, the shuttle vector comprises a recombinant insert.

In a more preferred embodiment of the shuttle vector said insert is part of a library.

In a most preferred embodiment said library is a cDNA library.

Advantageously, the above-described shuttle vector of the invention comprises at least one, such as two, three, four or five selectable markers. Selectable marker genes useful for the selection of transformed E. coli cells or transformed P. pastoris cells are well known to those skilled in the art and comprise, for example npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995), hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485) and amp^r which confers resistance to β-lactam antibiotics like ampicillin (Sykes and Mathew, J. Antimicrob. Chemother. 2: 115, 1976). Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

Among preferred selectable marker genes for use both in E. coli and P. pastoris is the bsd gene derived from Aspergillus terreus which encodes a blasticidin S deaminase and confers resistance to Blasticidin (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338) and the Sh ble gene (Streptoallteichus hindustanus bleomycin gene) which confers resistance to Zeocin™ (Calmes et al., Curr. Genet. 20, 309-314, 1991). The bsd gene as well as the Sh ble gene and the corresponding antibiotic selection agents are commercially available e.g. from Invitrogen Corporation, USA.

It is further preferred that the shuttle vector of the invention contains a selection marker functioning both in E. coli and P. pastoris. Preferably, said selection marker is the bsd gene or the Sh ble gene.

In another embodiment the present invention relates to a collection of shuttle vectors of the present invention.

The term "collection of shuttle vectors" denotes a plurality of shuttle vectors of the invention that comprise the same, the same and different, or different recombinant inserts. A collection of shuttle vectors may, e.g., be obtained by preparing a cDNA library and cloning the synthesized cDNAs or cDNA fragments into the shuttle vector of the invention. The present invention also relates to a host cell transformed/transfected with the shuttle vector of the present invention.

Methods for introducing nucleic acid molecules into cells are well known in the art and vary depending on the type of host used. For example, calcium chloride transfection is commonly utilized for prokaryotic cells whereas, e.g., calcium phosphate, liposome or DEAE-Dextran mediated transfection or electroporation may be used for eukaryotic host cells (see, e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y., and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989)). Electroporation is another method well-known in the art, for transfection in prokaryotic cells (Sambrook et al., 1989).

In a still further embodiment the present invention relates to a collection of host cells comprising the collection of shuttle vectors of the present invention. The term "collection of host cells" refers to at least two host cells, preferably a variety and most preferably a library of host cells that comprise the shuttle vector of the invention with the same, the same and different, or different recombinant inserts.

In a preferred embodiment of the present invention the host cell or the collection of host cells is/are Pichia pastoris cell(s).

In a further preferred embodiment of the present invention the host cell or the collection of host cells is/are E. coli cell(s).

In another embodiment the present invention relates to a method of producing a (poly)peptide comprising culturing the host cell of the present invention under suitable conditions and isolating said (poly)peptide from the culture. As will be readily appreciated by the person skilled in the art, depending on the expression system used, the synthesized fusion protein will be secreted into the culture medium or will accumulate in the cells. Thus, the term "culture" as used in accordance with the present invention comprises the culture medium and/or the cells used to synthesize the (poly)peptide of the invention. Suitable culture media, culture conditions like, e.g., temperature, and protocols for the recovery of (poly)peptides from the culture medium or from cells, including the disruption or lysis of the cells, are well known to the person skilled in the art (see, e.g., Sambrook et al., loc. cit. and Ausubel et al., loc. cit.).

If the (poly)peptide is to be secreted into the culture medium, sequences encoding a leader peptide, e.g, an alpha leader (for secretion in yeast) and/or, e.g., an ompA leader (for secretion in prokaryotes) may be cloned into the vector of the invention downstream of the T7 promoter. In addition, a sequence encoding a protease recognition sequence (e.g. for factor X or thrombin) may be cloned into the vector downstream of the leader sequences. This may be advantageously used for cleaving off the leader sequences after secretion of the (poly)peptide.

In yet another embodiment the present invention relates to a method of rearraying clones comprising picking the collection of host cells of the present invention; and transferring said collection of host cells in arrayed form to a liquid medium or a solid support.

The term "arrayed form" as used herein refers to any regular or non-regular form that can be replicated. Preferred are regular forms.

In a preferred embodiment, said arrayed form is a grid form. The grid should preferably allow for the high density array of clones. Suitable grids envisaged in accordance with the present invention are, e.g., a microtitre plate such as a 24 well, a 96 well or a 384 well microtitre plate (standard format: 13cmx8.5cm), a silica wafer, a chip (for example in the standard microscope slide format of 7.5cmx2.5cm), a mass spectrometry target or a matrix as described, e.g., in WO 99/57311 or WO 99/57312. In another preferred embodiment of the method of the present invention said picking and/or transferring is assisted or effected by automation.

Such automated devices may comprise a picking robot and/or spotting robot and/or gridding robot as reviewed in Cahill, 2000.

In a further preferred embodiment, the method further comprises the expression of the recombinant inserts comprised in the shuttle vector contained in the collection of host cells. In another embodiment the present invention relates to an array of clones that is obtainable by the method of the present invention.

Finally, the present invention relates to a kit comprising the vector; the collection of shuttle vectors; the host cell; the collection of host cells, and/or the array of the present invention, in one or more containers.

Suitable containers may be, e.g., vials and the components may, optionally, be. contained in buffers and/or solutions. Additionally or alternatively, one or more of said components may be adsorbed to a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtitre-plate, a glass slide, a mass spectrometry matrix, a BIAcore chip, a SPR chip, a gel, a coated surface, a gel coated surface, a porous surface, a non-porous surface, a teflon coated surface, a gold coated surface, or a mixture of surfaces.

The specification recites a number of documents. The disclosure content of said documents is herewith incorporated by reference.

The Figures show:

Figure 1 : The dual expression vector pZPARS-T7-RGSHis₆HA, that allows the dual expression in P. pastoris and E. coli.

Figure 2: Comparison of protein expression in P. pastoris and E. coli using the dual expression vector.

Proteins were expressed in P. pastoris and E. coli and purified under native and denatured conditions. The purified proteins were separated on SDS-PAGE on 12% polyacrylamide gels, electroblotted and detected by immunohistochemie as described in the examples. Lane A: native purified proteins expressed in P. pastoris; lane B: native purified protein expressed by E. coli; lane C: denatured purified proteins expressed by E. coli. I: 1 : human p47 (8); 2: EEF1A1 (20); 3: glucocorticoid receptor-associated protein (16); 4: splicing factor (27); 5: GAPDH (29); II: 1 unknown (26); 2: FEZ1 (14); 3: stathmin (18); 4: Zfp106 (11); 5: α-tubuline (4); III: 1 unknown (6); 2: cDNA DKFZp586J0619 (5); 3: RPL4 (1); 4: α-tubuline (7); 5 ribosomal protein (25); IV: 1: HSP90 (heat shock protein-90) (12); 2: PGD (10); 3: YWHAZ (13); 4: HSP90 (15); UCHL1 (21) (abbreviations as generally used as Genbank, public DNA and protein database entry names).

Figure 3: Expression in P. pastoris on a 24-well microtitre plate scale. For high-throughput scale expression ten clones were expressed in 24-well microtitre plate and purified as described in the examples. The eluates were separated by SDS-PAGE on a 12% polyacrylamide gel, electroblotted and detected by immunohistochemistry. Lane 1: GAPDH (29); lane 2: HSP90 (12); lane 3: EEF1A1 (9); lane 4: unknown (3); lane 5: human ribosomal protein (25); lane 6: FEZ1 (14); lane 7: unknown (6); lane 8: EEF1A1 (20); lane 9: unknown (clone 26); lane 10: human p47 (8) (abbreviations as generally used as Genbank, public DNA and protein database entry names).

Figure 4: GAPDH expression purified by GST-Sepharose-Beads A gel comparing expression of human GAPDH in pZPARST732-Cup-NST-BT having been induced with 100mM CuSo₄, and comparing expression in yeast strains GS115 and SMD1168 (a protease deficient strain). The results show that improved protein expression is obtained in the GS115 strain. Also various amounts of GST-Sepharose Beads were tested (as they are expensive) and from this experiment, the optimal amount is 100//I beads, when purifying from 1ml of cell lysate obtained from a 200ml culture volume.

The Examples illustrate the invention-

Example 1 : Strains, transformation and media

Escherichia coli strains XL-1 Blue and SCSI (Stratagene) were used for cloning and propagation of the recombinant plasmids and E. coli strain BL21(D3)pLysS (Invitrogen) was used as host for bacterial protein expression. Recombinant plasmids were transformed either by electroporation using a Gene Pulser (Bio-Rad) or by using rubidium chloride competent cells. E. coli transformants were selected on LB medium (0,5% yeast extract, 1% NaCI, 1% bactotryptone) containing 2% dextrose supplemented with 25 /g/ml zeozin. For selection and growth of BL21 (D3)pLysS, the medium contains additionally 34 μg/ml chloramphenicol. For protein expression, LB medium supplemented with 1 mM Isopropyl-b-D-thiogalactopyranosid (IPTG) was used. The Pichia pastoris: strain GS115 (his4, Mut+) (Invitrogen) was used for eukaryotic protein expression. The transformation of Pichia pastoris was performed by electroporation (Gene Pulser; Bio-Rad) according to the manufacturer's protocol but, using a resistance of 400 Ω instead of 200 Ω. Transformants were selected on YPD agar plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) supplemented with 100 μg/ml zeozin. Growth of Pichia pastoris cultures were performed in YPD medium supplemented with 100 or 120 μg/ml zeozin. For protein expression, Pichia pastoris cultures were transferred to BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB without amino acids, 0.5% methanol) supplemented with 100 μg/ml zeozin for induction by methanol.

Example 2: Plasmids

DNA isolation, restriction enzyme analysis, agarose-gel electrophoresis and cloning procedures were performed using standard techniques (Sambrook, 1989). All plasmid constructs were verified by DNA sequencing using the dideoxy-termination method (Sanger, 1977). For the Pichia pastoris shuttle vector, a 307 base pair T7- expression cassette was generated by PCR amplification from a modified pQE32 (Qiagen) with the oligonucleotides picT7-Shuttle5^'-35 (CGGTACGTAT TAATACGACT CACTATATTT GCTTTGTGAG CGGATAACAA TTA) and pQE276 (GGCAACCGAG CGTTCTGAAC), restricted with SnaBI/Notl and subcloned into pPIC3.5 (Invitrogen). The T7 expression cassette was transferred as a Avalll/Notl fragment into a modified pPICZa (Invitrogen) vector that was previously deleted for c- myc and 6x (His) tag sequences resulting in pZT7-RGSHisHA. The PARS1 sequence was amplified with the primers PARS1-5 (CTTGGATCCG ATAAGCTGGG GGAACATT) and PARS1-3 (TCCGGATCCA ATTAATATTT ACTTATTTTG GT) from the vector pYM8. The PCR product was restricted with BamHI and cloned into the BamHI-restricted and dephosphorilated pZT7-RGSHisHA, resulting in the final pZPARS-T7RGSHisHA vector. Example 3: cDNA expression library construction

For the construction of the cDNA library, the human fetal brain cDNA library, hExl , which was rearrayed based on its expression subset (Buessow, 1998), was arrayed on 2xYT-agar plates (230 mm x 230 mm; Genetics) and was grown for 16 h at 37 °C. The resulting colonies were scraped from the agar plates and the plasmid DNA was isolated. 10μg of hExl plasmid DNA was restricted using Sall/Notl. The DNA was electrophoretically separated and inserts in the range of 0.5 to 3 kb were isolated from the gel, purified (QIAGEN; QIAquick Gelextraction Kit), cloned into the Sall/Notl restricted pZPARS-T7RGSHisHA, transformed into E. coli (strain SCSI) plated out on 2xYT agar plates (230 mm x 230 mm; Genetics) and grown for 20 h at 37 °C. Approximately 100,000 colonies were scraped from the agar and plasmid DNA isolation was performed. This DNA preparation was used for transformation of P. pastoris.

Example 4: Sequence analysis

To prepare the clones for sequencing the P. pastoris clones were arrayed in a 96 well microtitre plate, each well was filled with 200 μl YPD supplemented with zeozin (100 μg/ml) and grown for three days at 30 °C. The inserts were amplified by PCR using the primers AOX5 (TTGCGACTGG TTCCAATTGA CAAG) and AOX3 (CATCTCTCAG GCAAATGGCA TTCTG). For PCR amplification cells of each clone was transferred twice using a 96-well Nunc replicator into an 50 μl reaction mix (50 mM KCI, 35 mM Tris-Base, 15 mM Tris-HCI, 1.5 mM MgCI, 0.1% Tween 20, 0.2 mM dWP^'s, 3 units Taq, 0.1 units lyticase (Sigma L2524)). The PCR was performed as follows: 30 min./37 °C; 4 min./94 °C; for 30 cycles: 45 seα/94 °C, 20 sec./ 55°C, 2.30 min.Λ72 °C; 10 min./72°C. The PCR products were purified and sequenced. Sequences analysis was performed using MacMolly Tetra (SoftGene GmbH) and by blasting against the public sequence databases (NCBI).

Example 5: Back transformation from P. pastoris into E. coli

To prepare plasmid DNA from P. pastoris clones 2 ml cultures of each expression clone were grown for 2 days at 30 °C. Cells were harvested by centrifugation at 2100g for 5 min. and protoplasts were prepared by shaking in 100 l lysis buffer (1 M sorbit, 10 mM sodium citrate pH 7.5, 10 mM EDTA, 5 mM DTT, 2 mg/ml lyticase (Seikagaku)) for 30 min. at 37 °C. Cells were lysed by the addition of 150 μl P2 buffer (Qiagen) and were incubated with shaking on ice for 30 min. After addition of 500 μl P3 buffer (Qiagen) the suspension was incubated on ice for 15 min. and centrifuged at 10,000 g for 10 min. The supernatant was purified (QIAquick Spin Miniprep Kit; QIAGEN) according to the manufacturer's protocol. The eluate was precipitated by ethanol, followed by resuspension of DNA in 10 μl Aqua dest. After dialysis against Aqua dest. 2-5 μl of DNA were used for transformation by electroporation in XH BIue and plated onto selective medium. XL-1Blue clones were confirmed by PCR and sequencing. Plasmid preparations were done from confirmed XL-1Blue clones (QIAquick Spin Miniprep Kit; QIAGEN) and 10 μl of DNA was transformed in BL21 (D3)pLysS by using the rubidium chloride method (Lueking et al., 2000).

Example 6: Protein expression

Escherichia coli: Proteins were expressed in E. coli (strain BL21 (D3)pLysS) liquid cultures. 2 ml LB medium containing 25 μg/ml zeozin and 34 μg/ml chloramphenicol were inoculated with 0.5 ml of an overnight culture and were shaken at 37 ^eC until an OD₆oo of 0.8- 1.0 was reached. IPTG was added to a final concentration of 1 mM. The culture was shaken for 5 h at 37 ^QC and cooled to 4 ^QC on ice. For native protein isolation, cells were harvested by centrifugation at 2,100 g for 5 min., washed in 1 ml phosphate buffer (50 mM NaH PO₄, 300 mM NaCI, pH 8.0). The cells were then lysed in 3 ml per gram wet weight of lysis buffer (50 mM Tris, 300 mM NaCI, pH 8.0) containing 10 mM imidazol, 1 mM PMSF, 0.25 mg/ml lysozyme, 1 mg/ml RNAse and 1 mg/ml DNAse at 4° C over night. The lysate was cleared by centrifugation at 10,000 g for 10 min. Ni-NTA agarose (Qiagen) was added and mixed by shaking at 4 ^SC for 1 h. The mixture was poured into a unpacked column and subsequently washed with ten bed volumes of lysis buffer containing 20 mM imidazole. Protein was eluted in lysis buffer containing 250 mM imidazole.

For denatured protein isolation, cells were harvested by centrifugation at 2,100 g for 5 min. and were washed in 1 ml phosphate buffer (50 mM NaH₂P0 , 300 mM NaCI, pH 8.0). The cells were then lysed in 500 μl buffer B (8 M urea, 100 mM NaH₂PO4, 10 mM Tris pH 8.0) for 30 min. at room temperature. Bacterial debris were pelleted by centrifugation at 10,000 g for 10 min. Ni-NTA agarose (QIAGEN) were added to the supematants and mixed by shaking at room temperature for 30 min. Ni-NTA agarose was then pelleted by centrifugation at 10,000 g for 5 min., washed with 1 ml buffer C (8 M urea, 100 mM NaH₂PO₄, 10 mM Tris pH 6.3) followed by elution of the proteins with 100μl buffer E (8 M urea, 100 mM NaH₂PO4, 10 mM Tris pH 4.5). Pichia pastoris: Proteins were expressed in P. pastoris (strain GS115) in liquid cultures. 20-100 ml BMMY medium (as described in Sambrook et al., 1989) was inoculated with 2 ml of a P. pastoris overnight culture grown in YPD and shaken at 30⁹C for 2-3 days and subsequently cooled to 4 ^QC on ice. For high-throughput scale expression clones were cultivated in 24-well microtitre plate with a culture volume of 5 ml. Cells were harvested by centrifugation at 2,100 g for 5 min, washed in 1 ml phosphate buffer (50 mM NaH₂PO₄, 300 mM NaCI, pH 8.0) and re-suspended in 0.3 ml lysis buffer (50 mM Tris, 300 mM NaCI, pH 8.0) containing 10 mM Imidazol, 1 mM PMSF. The cells were mixed with an equal volume of glass beads (size 0.5 mm), vortexed for 30 seconds and incubated on ice for 30 seconds. This procedure was repeated seven times and the lysate was cleared by centrifugation at 10,000 g for 5 min. Ni-NTA agarose (QIAGEN) was added and mixed by shaking at 4 ^QC for 1 h. The mixture was poured onto an unpacked column which was subsequently washed with ten bed volumes of lysis buffer containing 20 mM imidazole. Protein was eluted with lysis buffer containing 250 mM imidazole.

Example 7: Protein analysis

The purified proteins were separated by SDS/ PAGE (12.5 %) and transferred onto PVDF membrane (Millipore) by electroblotting (T77 SemiPhor, Pharmacia Biotech). After blotting, the filters were washed in TBST (TBS, 0.1 % (v/v) Tween 20) for 1 min, blocked in 2 % (w/v) bovine serum albumin (BSA)/ TBST for 60 min. and incubated with the monoclonal antibody α-RGSHis₆ (Qiagen) diluted 1 :2000 in 2 % (w/v) BSA/ TBST for 1 h at room temperature. This was followed by two 10 min. washes in TBST and a 1 h incubation with a goat α-mouse-HRP conjugated secondary antibody (Sigma) diluted 1 :5000 in 2 % (w/v) BSA/ TBST. Subsequently, the filters were washed in 20 ml TBST overnight and proteins were detected following incubation with western blot chemoluminiscence reagent (NEN). Protein concentrations were determined by BioRad assay according to the manufacturer's protocol. Example 8: Construction of a P. pastoris/ E. coli dual expression vector

The expression shuttle vector consists of the yeast alcohol oxidase (AOX) promoter, followed by the T7 promoter region including the bacterial ribosome binding site and a translation initiation site enabling transcription and expression in both hosts. For detection and purification of the target protein, the vector contains an amino-terminal oligo-histidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. By integration of a Pichia specific autonomous replicating sequence (PARS1), vector linearization is no longer required, increasing the transformation efficiency up to 10⁵ per μg DNA (Cregg, 1985). Furthermore, the PARS1 sequence allows the recovery of plasmids from yeast enabling a simple propagation from yeast to bacteria by transformation. Due to the use of a common selection marker zeozin, the size of the shuttle vector remains small resulting in convenient handling, cloning and transformation (Fig. 1).

Example 9: Construction and characterization of a human fetal brain (hExl) expression library in the dual expression vector

The hExl cDNA expression library (Buessow, 1998), which was re-arrayed based on its expression subset in E. coli, was sub-cloned into this novel dual expression vector and was then transformed into P. pastoris. 96 randomly chosen P. pastoris clones were picked and analyzed by PCR, DNA sequencing and protein expression. An average cDNA insert size of 1.6 kb was determined. 5 ^'-tag sequences of 95 clones were obtained and used for BLASTN searches against the public databases including Genbank and Unigene databases (Altschul, 1997). 83 (87 %) clones were found to match human proteins and 12 (13 %) clones were representing unknown sequences. 29 (30.2%) of the clones were fused to the N-terminal sequence MRGSHis₆ in the correct reading frame (RF+) and have subsequently revealed expression of heterologous protein. 9 (31 %) of them are full-length clones. Clones which do not contain an open reading frame (RF-) show no expression product. 24 (82.8 %) of the expression clones were shown to match a human protein. 5 (18 %) clones have revealed an unknown sequence. The molecular masses of proteins expressed from clones of known sequences were predicted by completing their 5'- tag sequence using the matching sequence in the database (Tab.1). Example 10: Comparison of protein expression in P. pastoris and E. coli.

Plasmids from the 29 P. pastoris expression clones were isolated and shuttled by transformation into E. coli. Expression products of all clones from both hosts were affinity-purified under native and denaturing conditions using Ni-NTA agarose. 29 (100 %) expression products were purified from P. pastoris in a soluble form. 25 (86.2 %) E. coli expressed proteins have been purified under denaturing conditions, whereas 8 (27.6%) proteins have been isolated in a soluble form, partly detected by sensitive detection methods, such as western immunoblotting (Fig. 2). Protein concentrations have been determined for each purified expression product and concentration in the range of 1.5-20 μg/ml or purified protein (denatured) were obtained from E. coli and in the range of 0.1 -5 μg/ml (native) when expressed in P. pastoris.

The protein sizes were determined by SDS-PAGE and the data were compared to the corresponding values predicted from DNA sequence data (Tab. 1). For the adaptation to high-throughput expression systems, we have expressed several proteins in P. pastoris in a 24-well microtitre plate, and have analyzed them by nickel chelate affinity purification and SDS-PAGE. Expression products could be detected for every clone tested (Fig.3).

Table 1 : Protein expression properties of the expression clones with the estimated protein weight.

Table 1

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Claims

1. A shuttle vector for expression of nucleic acid in Pichia pastoris and Escherichia coli comprising

(a) a promoter which is

(aa) a yeast alcohol oxidase (AOX) promoter;

(ab) a yeast CUS1 promoter;

(ac) a tetracycline promoter; or

(ad) a CMV promoter;

(b) an E. coli T7 promoter;

(c) a Pichia pastoris autonomously replicating sequence (PARS); and

(d) a multiple cloning site.

2. The shuttle vector of claim 1 further comprising

(e) a nucleic acid sequence encoding a tag.

3. The shuttle vector of claim 2, wherein said tag is an oligo-histidine domain (His 6), GST-tag or a hybrid tag comprising his-tag and biotin tag.

4. The shuttle vector of claim 2 or 3, wherein the tag is a part of a fusion protein upon expression of said nucleic acid.

5. The shuttle vector of claim 4, wherein said tag is positioned N-terminally of the fusion protein.

6. The shuttle vector of any one of claims 1 to 5, wherein said E. coli T7 promoter is placed downstream from said yeast AOX promoter.

7. The shuttle vector of any one of claims 1 to 6 comprising a recombinant insert.

8. The shuttle vector of claim 7, wherein said insert is part of a library.

9. The shuttle vector of claim 8, wherein said library is a cDNA library.

10. A collection of shuttle vectors of any one of claims 7 to 9.

11. A host cell transformed/transfected with the shuttle vector of any one of claims 1 to 9.

12. A collection of host cells comprising the collection of shuttle vectors of claim 10.

13. The host cell of claim 11 or the collection of host cells of claim 12, wherein said cell or cells is/are Pichia pastoris cell(s).

14. The host cell of claim 11 or the collection of host cells of claim 12, wherein said cell or cells is/are E. coli cell(s).

15. A method of producing a (poly)peptide comprising culturing the host cell of claim 13 or 14 under suitable conditions and isolating said (poly)peptide from the culture.

16. A method of rearraying clones comprising

(a) picking the collection of host cells of any one of claims 12 to 14; and

(b) transferring said collection of host cells in arrayed form to a liquid medium or a solid support.

17. The method of claim 16, wherein said picking and/or transferring is assisted or effected by automation.

18. The method of claim 16 or 17 further comprising the expression of the recombinant inserts comprised in the shuttle vector contained in the collection of host cells.

19. An array of clones obtainable by the method of claim 18.

0. A kit comprising

(a) the vector of any one of claims 1 to 9;

(b) the collection of shuttle vectors of claim 10;

(c) the host cell of claim 11 , 13 or 14;

(d) the collection of host cells of any one of claims 12 to 14, and/or

(e) the array of claim 19 or 20

in one or more containers.