Title of Invention

A METHOD FOR PRODUCING A HETEROLOGOUS PROTEIN

Abstract A heterologous protein is produced by means of a method in which yeast-type host cells are prepared, which, in addition to the at least one endogenous homologous DNA sequence coding for a first calnexin, comprise at least one recombinant DNA sequence coding for the protein and at least one additional recombinant DNA sequence coding for at least one second calnexin. The protein in the host cell represents a heterologous secretable protein. The host cells are induced to express the heterologous protein by means of the at least one sequence coding for the protein and to overexpress calnexins by means of the at least one additional sequence coding for the at least one second calnexin. The host cells are then induced to secrete the heterologous protein. The secreted heterologous protein is then separated off.
Full Text

Method for Production of a Heterologous Protein Using Yeast-Typehost Cells
The invention relates to a method for production of a heterologous protein, for which host cells of a yeast type are prepared that contain in each case at least one recombinant DNA sequence coding for the heterologous protein, then the host cells are caused to express and secrete the heterologous protein, and the secreted, heterologous protein is separated.
Methods of the type mentioned in the introduction are known, for example, from publication WO 00/68400 Al. The obtaining of proteins, which, for example, are used as active compounds in medications, with the help of recombinant DNA technology, has, among others, the advantage that the proteins can be made available in well characterised hosts in practically unlimited quantities. It is advantageous for the obtaining of recombinant proteins if, in each case, the expressed protein is secreted so that it can subsequently be obtained from the cell supernatant. This greatly simplifies the preparation, because the protein is already available in a relatively pure form and costly purification steps can be avoided. In many cases, yeasts are used in order to obtain secreted proteins on a large scale. These have the advantage that they can be kept relatively simply in cell cultures and lead to a good yield.
It is always the aim to increase the secretion of the heterologous proteins. In order to achieve this, it was proposed, for example, to increase the number of copies of the recombinant DNA sequences that code for the heterologous protein. Further, for an increase in yield of the protein, usually secreted in glycosylated form, it was proposed by Arima et al in 'Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae,' PEBS Letters 440 (1998), pages 89-92, to deactivate the gene of the yeast cells that code for the chaperone calnexin. Based on the results of this work, it can be expected that the calnexin reduces the secretion of heterologous proteins from yeast cells. In 'Calnexin Overexpression Increases Manganese Peroxidase Production in Asperigillus niger] in Applied and Environment Microbiology, February 2002, pages 846-851, Conesa et al describe that an overexpression of calnexin increases the production of manganese peroxidase; however, on the one hand, this applied to filamentary fungus cultures, and, on the other

hand, the positive influence of the calnexin overexpression on the yield was caused by the calnexin influencing heme incorporation into the manganese peroxidase.
The task of the invention is to provide a method by which the secretion of a heterologous protein is increased using yeast host cells. The task is solved by a method with the features of claim 1.
Using the method according to the invention for production of a protein, host cells were initially provided from at least the yeast strains of the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Pichia, Arxula, Schwanniomyces, Candida, or Yarrowia, which, along with their at least one own homologous DNA sequence coding for at least a first calnexin, they also contained in each case at least one recombinant DNA sequence coding for the protein, as well as at least one additional recombinant DNA sequence coding for a second calnexin, whereby the protein in the host cell represents a heterologous secretable protein. For example, the host cell contains a homologous DNA sequence, which codes for a first (own) calnexin. However, the host cells can also contain several of their own homologous DNA sequences, which code for a calnexin or also several different calnexins. According to the invention, the host cells to be prepared contain one or more additional recombinant DNA sequences, which code for at least a second calnexin. The terms 'first' and 'second' calnexin are used in the scope of this invention description to distinguish the homologous DNA sequence coded calnexin already present, on one hand, from the additional heterologous DNA sequence coded calnexins. However, the use of these terms alone does not imply a structural difference. The additional recombinant DNA sequences can comprise several different recombinant DNA sequences that code for the same or different (second) calnexins. Preferably, however, they are several copies of the same recombinant DNA sequence, which all code for the same second calnexin. Preferably, the second calnexin (the recombinant DNA sequence coding for that) is structurally similar or the same ('structurally similar' will be used here to mean that, on the one hand, codons that code for the same amino acid can be exchanged for one another, and, furthermore, that individual codons can be exchanged for codons that code for other amino acids, to the extent that the functions of the calnexin are thereby influenced at most insignificantly) as the first calnexin (coding for its own, homologous DNA sequence). A calnexin, in general, is a chaperone in the endoplasmic reticulum (ER) in any cell of an organism that, through binding on the oligosaccharide GlclMan9-GlcNAc2, is involved in the folding and quality

control of newly originating glycoproteins until these proteins are correctly folded or - because of an incorrect folding - are led to degradation. The term 'a calnexin' should - unless otherwise stated - include a calnexin from a yeast cell (CNE) as well as a calnexin (CNX) or calreticulin (CNR) from a mammalian cell or corresponding proteins from another cell.
The host cells are caused, with the help of at least one sequence coding for the protein, to express the heterologous protein, and with the help of at least one additional sequence coding for at least a second calnexin, to overexpress calnexins. Subsequently, the host cells are caused to secrete the heterologous protein. Finally, the secreted heterologous protein is separated.
In view of the state of the art described previously, the increasing effect of the overexpression of calnexins on the secretion of heterologous proteins in yeast cells is surprising. It is also surprising that a further increase of the protein secretion through calnexin overexpression has even been detected when the host cells already show a high yield of the secreted heterologous protein, even without calnexin overexpression.
In the preferred embodiment of the method according to the invention, during the host cell preparation step, at least one sequence coding for the heterologous protein and at least one additional DNA sequence coding for at least a second calnexin are inserted into the host cells with the help of vectors. Here, the vectors can be present in the cells as free plasmids. However, it is preferable for the particular vectors to be integrated into the genome of the cell.
The recombinant DNA sequences coding for the heterologous protein, on the one hand, and the additional recombinant DNA sequence coding for at least one second calnexin, on the other, can be present on the same vector. Preferably, however, every DNA sequence coded for the heterologous protein and every DNA sequence coding for a second calnexin is inserted on a corresponding separate vector. This simplifies the preparation of the vectors.
The host cells can be prepared in different ways. For example, recombinant host cells can be prepared initially that contain at least one recombinant DNA sequence coding for the heterologous protein, and the recombinant host cells can subsequently be supertransformed, in which in each

case at least one additional DNA sequence coding for a second calnexin is inserted into the recombinant host cells with the aid of vectors. Alternatively, the host cells could be prepared, whereby the recombinant host cells are first prepared that contain in each case at least one additional DNA sequence coding for at least a second calnexin, and the recombinant host cells can subsequently be supertransformed by insertion of in each case at least one recombinant DNA sequence coding for the heterologous protein in the recombinant host cells. Finally, it is also conceivable that during the step of preparing the host cells step at least one recombinant DNA sequence for the heterologous protein as well as at least one recombinant DNA sequence coding for at least a second calnexin are inserted into the host cell at the same time. For all three variants mentioned, appropriate expression plasmids are prepared as vectors before the step of preparing the host cells, whereby the vector in each case contains a DNA sequence coding for at least a second calnexin or a DNA sequence coding for the heterologous protein. The last-mentioned expression plasmids are prepared by isolating a calnexin gene (for example from the yeast type of host cells), and cloning it into a plasmid, whereby the calnexin gene is inserted between a homologous or a heterologous promoter and a homologous or heterologous terminator.
In a preferred embodiment of the method according to the invention, in step a) the host cells of a methylotrophic yeast are prepared, in particular a yeast of the genera Hansenula and Picia. Particularly preferable are host cells of the yeast Hansenula polymorpha.
In a preferred embodiment, the host cells of a recombinant yeast are prepared that contain in each case at least one recombinant DNA sequence coding for a heterologous protein, where the heterologous protein is a protein that during the expression and/or secretion is post-translationally modified, in particular post-translationally glycosylated. In doing so, the protein is a protein from a group that includes, for example, y-interferon, alginate epimerase, and consensus phytase.
In another embodiment, the heterologous protein is preferably a protein from a group that contains y-interferon, a-interferon, hirudin, serum albumins, alginate epimerase and consensus phytase.
Advantageous and/or preferable further developments of the method according to the invention are characterised in the dependent claims.

In the following, the invention is described in more detail using preferred embodiments. In the drawings:
Figure 1A and IB show the DNA sequences coding for the additional calnexin for insertion into the host cells (the appendixes 2A and 2B show the associated sequence listing);
Figure 2 shows a plasmid type of a calnexin expression plasmid, HCNE-Phleo, used for the supertransformation of a recombinant yeast host cell,
Figure 3 shows a plasmid type of a calnexin expression plasmid, TEFP-HCNE-Phleo, used in a recombinant yeast host cell, and
Figure 4 shows a Western blot of a culture supernatant of a strain of yeast cells that, according to the invention, cause the secretion of alginate C5 epimerase.
Initially, host cells of a yeast type are prepared that contain, along with their own homologous DNA sequence coding for calnexin, several copies of a recombinant DNA sequence coded for a heterologous protein, as well as several copies of an additional recombinant DNA sequence coding for calnexin. Preferably, recombinant Hansenula polymorpha host cells are prepared.
To producer the recombinant host cells, expression plasmids are first prepared as vectors. For this, the calnexin gene of the yeast Hansenula polymorpha, shown in Figure 1A and appendix 2A (the homologue of the calnexin gene CNE1 of Saccharomyces cerevisiae - cf Figure IB), from a Hansenula polymorpha strain with the name RBI 1 was isolated, cloned, and sequenced using a polymerase chain reaction.
In experiments, two expression plasmids were used that contained the calnexin gene of Hansenula polymorpha. In an expression plasmid with the name HCNE-Phleo (HCNE' derives from: Hansenula calnexin; 'Phleo' derives from genes resistant against the antibiotic selection phleomycin), the calnexin gene was flanked by its original transcription promoter and its original

terminator. This plasmid is illustrated in Figure 2. In the expression plasmid illustrated in Figure 3, TEFP-HCNE-Phleo (TEFP1 derives from transcriptase elongation factor promoter), the calnexin gene was controlled by the TEF1 promoter from the yeast Arxula adeninivorans and the MOX terminator from Hansenula polymorpha. The TEF promoter used in this vector to control CNE expression can be inserted as a universal element for expression in various yeast types (see Klabunde, J, Kunze, G, Gellissen, G, and Hollenberg, CP, 'Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-dzrived DNA-targeting element.' FEMS Yeast Research 4, 185-198, 2003). Both expression plasmids were subsequently used for supertransformation of three different recombinant strains of Hansenula polymorpha, which in each case secreted a heterologous protein. Of the three tested recombinant proteins, it was known that these were modified by a posttranslational glycosylisation in the host cells of the Hansenula polymorpha. The following recombinant strains of Hansenula polymorpha were chosen for the supertransformation:
a) RBll/AlgElsyn 52-4, which secretes a bacterial alginate C5 epimirase,
b) RBll/Conphys 3-68, which secretes a synthetic 'consensus' phytase,
c) RBI 1/FMD-MFTFND 23-2, which secretes a variant human y-interferon, which is over-glycosylated when Hansenula polymorpha is used a large extent, and
d) RB11/HSA 56-1, which secretes a human serum albumin.
Each of these strains was supertransformed with one of the two previously mentioned expression plasmids (HCNE-Phleo or TEFP-HCNE-Phleo). The supertransformants were selected with respect to resistance against the antibiotic phleomycin. After stabilisation, the supertransformants were cultivated using the conditions for heterologous protein expression, and the secreted heterologous proteins were analysed using Western blots or the SDS-PAGE/Coomassie stain. Figure 4 shows an example Western blot of the culture supernatant from a recombinant Hansenula polymorpha strain RBI 1/AlgElsyn 52-4, which expresses the bacterial alginate C5 epimerase and was supertransformed with the expression plasmid HCNE-Phleo (d) (track 3-8) or TEFP-HCNE (tracks 9-15). Track 1 shows an AlgEl Standard (E coli\ and track 2 shows a protein-size marker. Track 16 shows for comparison a non-supertransformed recombinant strain of RBI 1/AlgElsyn 52-4, and track 17 shows a control strain, which is not recombinant. The results of this analysis, in

particular Figure 4, clearly show an increased quantity of secreted proteins using the analysed supertranformants. This is true for both of the calnexin expression plasmids used.
The number of copies of the expression vectors in the three tested recombinant strains was unchanged, so the increase in the quantity of protein is not attributed to a gene dosage effect, but instead solely to the effect of the calnexin. The positive effect of the calnexin overexpression was detectable using strains with a reduced number of integrated foreign gene vectors (2 copies per cell; RBH/AlgElsyn 52-4), as well as using strains with a larger number of integrated foreign gene vectors (appx 50 copies per cell; RB11/FMD-MFIFNG 23-2 and RBll/Conphys 3-68). The positive effect of the calnexin overexpression was also detectable over the total range of the foreign protein production (a few milligrams of IFN gamma per litre in RB11/FMD-MFIFNG 23-2; 1-2 grams of epimerase per litre in RBll/AlgElsyn 52-4; 13.5 grams of phytase per litre in RBll/Conphys 3-68) in yeast. Additionally, for a more exact detection of the effect of the calnexin overexpression on the heterologous protein secretion, the HSA production in strain 56-1 and in selected calnexin supertransformants was quantified and compared using ELISA (see appendix 1). Furthermore, the strain RBI 1/HSA 56-1 was supertransformed with a vector without a calnexin gene, in order to provide evidence that the increase in production is attributed solely to the additional copies of calnexin and not to another vector sequences.
For this, four control strain vectors (mock HSA #1 to #4), which arose from the transformation of HSA 56-1 with the empty vector pPhleo(d), were tested in same way for their HSA expression capability.



The overexpression of calnexin as chaperone of the endoplasmatic reticulum using a recombinant strain of Hansenula polymorpha, which expresses and secretes a glycosylated heterologous protein, therefore leads to a significant improvement of the secretion efficiency and the yield of these heterologous proteins.
Materials and Methods:
The methods and media used for the construction of the expression vector, as well as for the transformation, cultivation, and analysis of the yeast cells, and also for the base vectors and yeast strains are described in:
G Gellissen (ed), 'Hansenula polymorpha - biology and applications', Wiley-VCH, Weinheim 2002, in particular, in the chapters:
A Degelmann, 'Methods' and
M Suckow, G Gellissen, 'The expression platform based on Hansenula polymorpha strain RBI 1 and its derivatives - history, status and perspectives.'

- Appendix 1 -ELISA Procedure:
Dilute the culture remainder with PBS (phosphate buffered saline) 1:1000 (1:500 to 1:200)
in uncoated micro titer plates (in 2 or 3 steps: first 1:20, then 1:25, and possibly, 1:50).
From the protein standard (serum-derived human albumin, Rockland Nr. 009-0133;
concentration 12.5 mg/mL) create serial dilutions in PBS. From the dilution steps 3 - 9,
50 (iL are accumulated, in each case, in duplicate.
Addition: In each case, pipette 50 )iL of the diluted remainder and standard dilutions into
the wells of a coated micro titer plate (Nunc Maxisorp Nr. 442404).
Cover the plates with adhesive film and incubate overnight at 4°C (without shaking).
Empty the plate contents into a washbasin, wash the wells 3x with PBST (PBS-Tween).
After the last wash step, remove the remaining fluid by tapping the micro titer plates on
paper towels (wells facing downwards).
Blocking: pipette 200 \iL of blocking solution into the wells; incubate for 1 hour at RT with
shaking.
Wash the wells 3x with PBST.
Bonding of the HSA (human serum albumin) antibody: dilute peroxidase conjugated IgG
fraction of anti-human albumin (rabbit; Rockland Nr. 209-4333) for long term storage 1:2
with glycerin. Dilute the parent solution (5 mg/mL) 1:500 in PBST, in each case, pipette 50
JIL into the wells; incubate for 1 hour at RT with shaking.
Wash the wells 3x with PBST.
Development: Mix 1 Vol. TMB (tetramethyl benzidine) peroxidase substrate and 1 Vol.
peroxidase solution B (KPL Nr. 50-76-00); pipette 100 ^L into each well. Agitate at RT,
until the desired color intensity appears, then stop the development through the addition of
100 pL 1M phosphoric acid.
Finally, measure the absorption at 450nm in the plate reader.

Solutions: 10 x PBS 1.37 M NaCl 27 mM KC1 43 mM Na2HP04 14 mM KH2P04
PBST
PBS/ 0.05% Tween 20
Blocking solution
1% skimmed milk powder in PBS
Peroxidase substrate: 3, 3', 5, 5'-tetramethyl benzidine


Claims
1. A process for producing a protein, where:
a) host cells are prepared from at least one yeast type of genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Pichia, Arxula, Schwanniomyces, Candida, and Yarrowia, which along with their at least one, own, homologous DNA sequence coding for at least a first calnexin, also contain in each case, at least one recombinant DNA sequence coding for the protein, as well as at least one additional recombinant DNA sequence coding for a second calnexin, where the protein in the host cell represents a heterologous, secretable protein;
b) the host cells are caused, with the help of at least one sequence coding for the protein, to express the heterologous proteins, and, with the help of at least one additional sequence coding for at least a second calnexin, to overexpress calnexins;
c) the host cells are caused to secrete the heterologous protein, and
d) the secreted heterologous protein is separated.

2. The process according to claim 1, characterised in that in step a), host cells are prepared that contain several additional recombinant DNA sequences coding for a second calnexin.
3. The process according to claim 2, characterised in that the several additional recombinant DNA sequences are identical to one another.
4. The process according to one of claims 1 - 3, characterised in that the first and the second calnexins are structurally similar, preferably identical to each other.
5. The process according to claim 4, characterised in that the additional recombinant DNA sequences coding for calnexin are similar to one of the homologous DNA sequences coding for calnexin, preferably identical.
6. The process according to one of the claims 1-5, characterised in that all recombinant DNA sequences coding for the heterologous protein are identical.

7. The process according to the claims 1-6, characterised in that the step of preparing host cells, at least one DNA sequence coding for the heterologous protein and at least one additional DNA sequence coding for a second calnexin are inserted into the host cell with the aid of vectors.
8. The process according to claim 7, characterised in that the vectors are integrated into the genome of the cell.
9. The process according to claim 7 or 8, characterised in that each DNA sequence coding for the heterologous protein and each DNA sequence coding for a second calnexin are each inserted into a vector.

10. The process according to one of the claims 7- 9, characterised in that plasmids are used as vectors.
11. The process according to one of claims 1-10, characterised in that during the step of preparing the host cells,
al) recombinant host cells are first prepared that contain in each case at least one recombinant DNA sequence coding for a heterologous protein, and
a2) subsequently, the recombinant host cells are supertransformed by inserting into the recombinant host cells at least one additional DNA sequence coding in each case for at least a second calnexin, with the aid of vectors.
12. The process according to one of claims 1-10, characterised in that during the step of preparing
the host cells,
al) recombinant host cells are first prepared that contain in each case at least one additional recombinant DNA sequence coding for a second calnexin, and
a2) the recombinant host cells are subsequently supertransformed by inserting into the recombinant host cells at least one recombinant DNA sequence coding in each case for the heterologous protein.
13. The process according to the claims 1-10, characterised in that the step of preparing the host

cells, at least one recombinant DNA sequence coding for the heterologous protein as well as at least one recombinant DNA sequence coding for at least a second calnexin are inserted into the host cell at the same time.
14. The process according to one of the claims 11-13, characterised in that before step a) expression plasmids are prepared as vectors that contain a DNA sequence for at least a second calnexin or a DNA sequence coding for the heterologous protein.
15. The process according to claim 14, characterised in that the expression plasmids are prepared, whereby a calnexin gene is isolated and cloned into a plasmid, where the calnexin gene is inserted between a homologous or heterologous promoter and a homologous or heterologous terminator.
16. The process according to one of the claims 1-15, characterised in that in step a), host cells are prepared from a methylothropheriic yeast, in particular of the genera Hansenula and Pichia.
17. The process according to claim 16, characterised in that in step a), host cells are prepared from the yeast Hansenula polymorpha.
18. The process according to one of the claims 1-17, characterised in that in step a), host cells are prepared from a recombinant yeast, which in each case contain at least one recombinant DNA sequence coding for a heterologous protein, where the heterologous protein is a protein that is post-translationally modified during step b) and/or step c).
19. The process according to claim 18, characterised in that the heterologous protein is a protein that is post-translationally glycosylated.
20. The process according to claim 19, characterised in that the heterologous protein is a protein from a group that includes y-interferon, alginate epimerase, and consensus phytase and variants of y-interferon, alginate epimerase, and consensus phytase.
21. The process according to claims 1 to 17, characterised in that the heterologous protein is a

protein from a group that includes y-interferon, a-interferon, hirudin, serum albumin, alginate epimerase, consensus phytase, and variants of y-interferon, a-interferon, hirudin, serum albumin, alginate epimerase, and consensus phytase.


Documents:

2118-CHENP-2007 AMENDED CLAIMS 02-11-2010.pdf

2118-CHENP-2007 AMENDED PAGES OF SPECIFICATION 02-11-2010.pdf

2118-chenp-2007 form-3 02-11-2010.pdf

2118-CHENP-2007 CORRESPONDENCE OTHERS 20-05-2010.pdf

2118-CHENP-2007 EXAMINATION REPORT REPLY RECIEVED 02-11-2010.pdf

2118-CHENP-2007 POWER OF ATTORNEY 02-11-2010.pdf

2118-chenp-2007-abstract.pdf

2118-chenp-2007-claims.pdf

2118-chenp-2007-correspondnece-others.pdf

2118-chenp-2007-description(complete).pdf

2118-chenp-2007-drawings.pdf

2118-chenp-2007-form 1.pdf

2118-chenp-2007-form 3.pdf

2118-chenp-2007-form 5.pdf

2118-chenp-2007-pct.pdf


Patent Number 247268
Indian Patent Application Number 2118/CHENP/2007
PG Journal Number 13/2011
Publication Date 01-Apr-2011
Grant Date 30-Mar-2011
Date of Filing 17-May-2007
Name of Patentee ARTES BIOTECHNOLOGY GMBH
Applicant Address AGNESSTRASSE 8, 45136 ESSEN.
Inventors:
# Inventor's Name Inventor's Address
1 GELLISSEN, GERD RINGSTRASSE 30, 42489 WULFRATH.
2 KLABUNDE, JENS KAROLINGERSTRASSE 37, 40223 DUSSELDORF, GERMANY
3 DEGELMANN, ADELHEID PAULSMUHLENSTRASSE 64, 40597 DUSSELDORF, GERMANY
4 HOLLENBERG, CORNELIS MANNESMANNUFER 5, 40213 DUSSELDORF, GERMANY
PCT International Classification Number C12N 1/19
PCT International Application Number PCT/EP05/11942
PCT International Filing date 2005-11-08
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04027280.9 2004-11-17 EUROPEAN UNION