Mating factor alpha propeptide variants

Abstract

The present invention relates to mating factor alpha original peptide variants. The present invention relates to mating factor alpha propeptide variants useful for the recombinant expression of polypeptides comprising a GLP-1 peptide in yeast. The invention also relates to DNA sequences, vectors and host cells for expressing polypeptides in yeast.

Description

[0001] This application is a divisional application. The filing date of the original application was March 2, 2015, the application number was 201580010737.4 (PCT / EP2015 / 054298), and the invention title was "Mating Factor Alpha Propeptide Variants". Technical Field

[0002] The present invention relates to the technical fields of protein expression and protein chemistry, in which polypeptides are prepared by recombinant expression in yeast. Background Art

[0003] Recombinant polypeptide expression techniques enable the production of large amounts of desired polypeptides that are useful, for example, due to their biological activity. Such polypeptides are typically expressed as recombinant fusion polypeptides in microbial host cells. The polypeptide of interest is often linked to a fusion partner polypeptide in order to increase the expression level, facilitate secretion, increase solubility, promote polypeptide folding, protect the polypeptide from unintended proteolysis, or facilitate the purification of the polypeptide of interest.

[0004] To ensure the secretion of recombinantly expressed polypeptides from yeast, a pre-pro peptide (commonly referred to as a "leader sequence") is typically fused to the N-terminus of the recombinant product. This pre-sequence ensures the translocation of the fusion protein into the endoplasmic reticulum (ER), which is the starting point of the secretory pathway. This pro-sequence ensures further transport from the ER to the Golgi apparatus, where the endogenous protease known as Kex2p is typically used to cleave off the pro-sequence. The processed recombinant peptide is then secreted into the growth medium from which the recombinant peptide can be purified.

[0005] When secreting recombinant polypeptides, the pre-pro sequence from Mating Factor Alpha is typically used as the leader sequence. However, many other sequences are capable of facilitating the secretion process. The leader sequence has an important influence not only on the amount of the secreted peptide, but also on the quality with respect to degradation and post-translational modifications such as O-glycosylation. Since degradation and O-glycosylation are typically undesired events, a leader sequence that minimizes these modifications and at the same time maximizes the yield of the secreted polypeptide is desired.

[0006] EP 0121884 A2 describes the recombinant production of human insulin in Saccharomyces cerevisiae using yeast α-factor.

[0007] EP 0324274 A1 describes the use of a truncated α-factor leader sequence to improve the expression and secretion of heterologous proteins in yeast.

[0008] Thim et al. (PNAS 83 (1986) 6766 - 6770) described the secretion and processing of pro - insulin in Saccharomyces cerevisiae using mating factor α.

[0009] WO95 / 34666 described synthetic leader sequences for the production of secreted polypeptides in Saccharomyces cerevisiae.

[0010] Rakestraw et al. (Biotech. Bioeng. 103 (2009) 1192 - 1201) described a mutated mating factor α leader sequence that increases the secretion of single - chain antibodies and the production level of human IgG1 in Saccharomyces cerevisiae.

[0011] There is a need for more specific leader sequences that increase the yield of polypeptide precursors and reduce the proportion of O - glycosylated recombinant polypeptides. In particular, there is a need for a leader sequence for expressing the GLP - 1 peptide in yeast that increases the yield of the GLP - 1 peptide or its precursor and reduces the O - glycosylation of the GLP - 1 peptide. Such more specific leader sequences can facilitate higher yields of recombinant polypeptides and lower amounts of O - glycosylated impurities. Summary of the Invention

[0012] One object of the present invention is to provide yeast cells with an increased level of heterologous polypeptide expression. Another object of the present invention is to provide yeast cells that secrete a recombinant polypeptide having a reduced amount of O - glycosylated variants of the recombinant polypeptide. In particular, one object of the present invention is to provide yeast cells having an increased level of recombinant polypeptide expression and a reduced amount of O - glycosylated variants. One object of the present invention is to provide an improved expression system for the recombinant expression of the GLP - 1 peptide in yeast cells.

[0013] According to a first aspect of the present invention, there is provided a method for the recombinant expression in yeast of a polypeptide comprising a GLP - 1 peptide, which comprises culturing a yeast strain comprising a DNA sequence encoding a processing and secretion signal upstream of the polypeptide, wherein the processing and secretion signal comprises a mating factor α pro - peptide variant having at least one substitution in the VIGYL sequence at positions 38 - 42 to comprise an amino acid sequence of general formula (I):

[0014] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0015] Wherein

[0016] X 38 is F, L, I or V;

[0017] X39 is L, I, V or M;

[0018] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0019] X 41 is S, Y, F, W, L, I, V or M;

[0020] X 42 is Y, W, L, I, V, M or S;

[0021] with the condition that X 38 -X 42 is not VIGYS.

[0022] In one embodiment of a method for recombinant expression of a polypeptide in yeast, the mating factor α propeptide variant has at least one substitution in the VIGYL sequence at positions 38 - 42, thereby comprising an amino acid sequence of general formula (I):

[0023] X 38 -X 39 -X 40 -X 41 -X 42 (I)

[0024] wherein X 38 is V; X 39 is L, I, V or M; X 40 is G or R; X 41 is Y, and X 42 is L.

[0025] In another embodiment, the polypeptide for recombinant expression is a GLP - 1 peptide.

[0026] In another embodiment, the polypeptide for recombinant expression comprises GLP - 1(7 - 37)[K34R], GLP - 1(9 - 37)[K34R] or GLP - 1(9 - 37)[K34R, G37K].

[0027] According to a second aspect of the present invention, there is provided a mating factor α propeptide variant which has at least one substitution in the VIGYL sequence at positions 38 - 42, thereby comprising an amino acid sequence of general formula (I):

[0028] X 38 -X 39 -X 40 -X 41 -X 42 (I)

[0029] wherein

[0030] X 38 is F, L, I or V;

[0031] X 39 is L, I, V or M;

[0032] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0033] X 41 is S, Y, F, W, L, I, V or M;

[0034] X 42 is Y, W, L, I, V, M or S;

[0035] Provided that X 38 -X 42 is not VIGYS.

[0036] According to the third aspect of the present invention, there is provided a GLP-1 precursor, which is a fusion polypeptide comprising:

[0037] - a pre-peptide,

[0038] - a mating factor alpha propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42, thereby comprising an amino acid sequence of general formula (I):

[0039] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0040] wherein

[0041] X 38 is F, L, I or V;

[0042] X 39 is L, I, V or M;

[0043] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0044] X 41 is S, Y, F, W, L, I, V or M;

[0045] X 42 is Y, W, L, I, V, M or S;

[0046] Provided that X 38 -X 42Not VIGYS,

[0047] - An optional extension peptide, and

[0048] - A GLP-1 polypeptide.

[0049] According to the fourth aspect of the present invention, there is provided a DNA sequence encoding the mating factor α propeptide variant or GLP-1 precursor.

[0050] According to the fifth aspect of the present invention, there is provided an expression vector comprising a DNA sequence encoding the mating factor α propeptide variant or GLP-1 precursor.

[0051] According to the sixth aspect of the present invention, there is provided a host cell comprising the expression vector according to the present invention.

[0052] In one embodiment, the host cell for expression has a non-functional pmt1 gene or no pmt1 gene at all. It has surprisingly been found that such host cells reduce the amount of O-glycosylated polypeptide independently of the reduction in the amount of O-glycosylated polypeptide obtained from the mating factor α propeptide variant of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows the minimal expression plasmid used in Example 1.

[0054] Figure 2 Shows the N-terminally extended GLP-1(7-37)[K34R] precursor, which includes the wild-type mating factor α prepropeptide, indicating the VIGYL subsequence of the propeptide. DETAILED DESCRIPTION

[0055] According to the first aspect of the present invention, there is provided a method for recombinant expression of a polypeptide in yeast, which comprises culturing a yeast strain comprising a DNA sequence encoding a processing and secretion signal upstream of the polypeptide, wherein the processing and secretion signal comprises a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42 to comprise an amino acid sequence of the general formula (I):

[0056] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0057] Wherein

[0058] X 38 is F, L, I or V;

[0059] X39 is L, I, V or M;

[0060] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0061] X 41 is S, Y, F, W, L, I, V or M;

[0062] X 42 is Y, W, L, I, V, M or S;

[0063] provided that X 38 -X 42 is not VIGYS.

[0064] As used herein, the term "leader sequence" is intended to mean an amino acid sequence consisting of a prepeptide (signal peptide) and a propeptide. Non-limiting examples of leader sequences are, for example, the α-factor signal leader sequence from Saccharomyces cerevisiae and the synthetic leader sequences for yeast as described in WO95 / 34666.

[0065] As used herein, "prepeptide" is intended to mean a signal peptide present as an N-terminal sequence on the precursor form of a polypeptide to be expressed. The function of the signal peptide is to facilitate translocation of the polypeptide into the endoplasmic reticulum in a host cell. The signal peptide is typically cleaved off during this process. The signal peptide can be heterologous or homologous to the host cell producing the polypeptide.

[0066] As used herein, "propeptide" is intended to mean a peptide sequence whose function is to direct the expressed polypeptide from the endoplasmic reticulum to the Golgi apparatus and further to secretory vesicles for secretion into the culture medium (i.e., the polypeptide is exported across the cell wall or at least through the cell membrane into the periplasmic space of the yeast cell). Non-limiting examples of propeptides are the yeast α-factor propeptide (see US4,546,082 and 4,870,008) and the synthetic propeptides disclosed in US 5,395,922, 5,795,746, 5,162,498 and WO 98 / 32867. The propeptide will preferably contain an endopeptidase processing site at the C-terminus, such as the Lys-Arg sequence or any functional analogue thereof.

[0067] As used herein, the term "mating factor α" (MFα, MFa or MFalpha) is intended to mean the Saccharomyces cerevisiae prepro sequence which contains the mating factor α prepeptide as amino acid residues 1-19 and the mating factor α propeptide as amino acid residues 20-85 in the structure MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVA VLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR (SEQ ID NO:2).

[0068] The mating factor α contains the sequence VIGYL (SEQ ID NO:3) as amino acid residues 38 - 42. Variants of the mating factor α have been used for recombinant expression of polypeptides, which contain the sequence VIGYS (SEQ ID NO:4) as amino acid residues 38 - 42 in the mating factor α sequence.

[0069] As used herein, the terms “polypeptide”, “protein” and “peptide” may be used interchangeably to denote a polypeptide. It should be understood that the specific terms used place no limitation on the size of the molecule (unless stated directly in a particular instance).

[0070] Amino acid residues are usually designated according to the single - letter abbreviations in accordance with the IUPAC nomenclature, e.g., D denotes aspartic acid (Asp) and G denotes glycine. However, in some cases the corresponding three - letter abbreviations are also used.

[0071] “Genetically - encoded amino acids” as used herein are intended to denote the group consisting of: G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S, T and any of their biological modifications. Non - limiting examples of such biological modifications are, for example, amidation, glycosylation and disulfide bond formation.

[0072] “Analogue” as used herein is intended to denote a polypeptide derived from a reference polypeptide by substitution, deletion and / or addition of one or more amino acid residues from the reference polypeptide. Non - limiting examples of analogues of GLP - 1(7 - 37) (SEQ ID NO:5) are GLP - 1(7 - 37)[K34R] (SEQ ID NO:6) in which residue 34 has been replaced by an arginine residue and GLP - 1(9 - 37)[K34R] (SEQ ID NO:7) in which residue 34 has been replaced by an arginine residue and amino acid residues 7 - 8 have been deleted (using the common numbering for amino acid residues of the GLP - 1 peptide).

[0073] “Variant” with respect to a polypeptide as used herein is intended to denote a chemical variant of the polypeptide that retains substantially the same primary function as the original protein. Thus, a variant is generally a modified form of the polypeptide in which as few modifications as possible are introduced that confer some desired property on the modified polypeptide while retaining substantially the same primary function of the original polypeptide. Non - limiting examples of polypeptide variants are, for example, extended polypeptides, truncated polypeptides, fusion polypeptides and analogues. A non - limiting example of a variant of the mating factor α propeptide is L42S - mating factor α(20 - 85) (SEQ ID NO:8). A non - limiting example of a variant of GLP - 1(7 - 37) is GLP - 1(7 - 37)[K34R].

[0074] In one embodiment, the variant of the polypeptide contains 1-2 amino acid substitutions, deletions or additions as compared to the unmodified polypeptide. In another embodiment, the variant contains 1-5 amino acid substitutions, deletions or additions as compared to the unmodified polypeptide. In another embodiment, the variant contains 1-15 amino acid substitutions, deletions or additions relative to the corresponding unmodified polypeptide.

[0075] According to a second aspect of the present invention, there is provided a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42, thereby comprising an amino acid sequence of general formula (I):

[0076] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0077] wherein

[0078] X 38 is F, L, I or V;

[0079] X 39 is L, I, V or M;

[0080] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0081] X 41 is S, Y, F, W, L, I, V or M;

[0082] X 42 is Y, W, L, I, V, M or S;

[0083] provided that X 38 -X 42 is not VIGYS. In one embodiment, the mating factor α propeptide variant does not contain VIGYS, VIDYS, VATYL, VIGYR or AIGYL as X 38 -X 42 .

[0084] According to a third aspect of the present invention, there is provided a GLP-1 precursor, which is a fusion polypeptide comprising:

[0085] - a propeptide,

[0086] - a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42, thereby comprising an amino acid sequence of general formula (I):

[0087] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0088] wherein

[0089] X 38 is F, L, I or V;

[0090] X 39 is L, I, V or M;

[0091] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0092] X 41 is S, Y, F, W, L, I, V or M;

[0093] X 42 is Y, W, L, I, V, M or S;

[0094] provided that X 38 -X 42 is not VIGYS,

[0095] - an optional extension peptide, and

[0096] - a GLP-1 peptide.

[0097] As used herein, the term "GLP-1 peptide" is intended to mean GLP-1(7-37), GLP-1(7-36) amide and their analogs, which can be produced by conventional recombinant DNA techniques as well as conventional synthetic methods. Such GLP-1 peptides include, but are not limited to, native glucagon-like peptide-1, for example, such peptide fragments containing GLP-1(7-37) and its functional variants as disclosed in WO 87 / 06941; such peptide fragments containing GLP-1(7-36) and its functional derivatives as disclosed in WO 90 / 11296; such analogs of the active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37 as disclosed in WO 91 / 11457; such N-terminal truncated fragments of GLP-1 as disclosed in EP0699686-A2; and such GLP-1 analogs and derivatives containing an N-terminal imidazole group as disclosed in EP 0708179-A2. Non-limiting examples of GLP-1 peptides are GLP-1(7-37) and GLP-1(7-37)[K34R].

[0098] As used herein, the term "GLP-1 precursor" is intended to mean a polypeptide comprising an extended GLP-1 peptide, wherein the extension is used to promote the secretion, expression, or recovery of the GLP-1 peptide. Examples of GLP-1 precursors can be found in WO03 / 010186 and WO09 / 083549. The GLP-1 precursor is intended to include GLP-1 peptides having a small extension, such as 2-5 amino acid residues, as well as GLP-1 peptides having a longer extension comprising a propeptide and a pro-propeptide.

[0099] In one embodiment of the method for recombinant expression of a polypeptide in yeast, the amino acid sequence of the general formula (I) has a sequence wherein

[0100] X 38 is F, L or V;

[0101] X 39 is L, I, V or M;

[0102] X 40 is G or R;

[0103] X 41 is S, Y, L, I, V or M, and

[0104] X 42 is Y, W, L, V or M.

[0105] In another embodiment, the amino acid sequence of the general formula (I) has a sequence wherein

[0106] X 38 is I or V;

[0107] X 39 is L, I, V or M;

[0108] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0109] X 41 is Y, F or W, and

[0110] X 42 is L or I.

[0111] In another embodiment, the amino acid sequence of the general formula (I) has a sequence wherein

[0112] X 38 is V;

[0113] X 39 is L, I, V or M;

[0114] X 40 is G or R;

[0115] X 41 is Y, and

[0116] X 42 is L.

[0117] In another embodiment, the amino acid sequence of formula (I) has a sequence in which X 40 is R. In another embodiment, the amino acid sequence of formula (I) has a sequence in which X 40 is R, and X 42 is L.

[0118] In yet another embodiment, the amino acid sequence of formula (I) has a sequence in which X 38 is V, X 39 is I, X 40 is R, X 41 is Y, and X 42 is L.

[0119] In another embodiment, the recombinantly expressed polypeptide is a GLP-1 peptide or a variant thereof, such as GLP-1(7-37)[K34R] or GLP-1(9-37)[K34R].

[0120] In one embodiment, the recombinantly expressed polypeptide has an N-terminal extension, i.e., located between the mating factor α variant and the polypeptide to be produced. This extension can facilitate the expression or secretion of the polypeptide by the host cell, or it can protect a portion of the polypeptide from unwanted proteolytic processing at the N-terminus. In another embodiment, the N-terminal extension is a polypeptide having 2-10 amino acid residues or having about 8 to about 200 amino acid residues. When the extension is used to facilitate the expression of the polypeptide in the host cell, or when the extension is used to protect the polypeptide from proteolytic processing at the N-terminus, a smaller N-terminal extension is typically used. In another embodiment, the N-terminal extension is selected from EEK, EEAEK, HK, EEAHK, E(EA)2HK, E(EA)3HK, EEGHK, EHPK, EEGEPK, EEAHELK, EEAHEVK, EEAHEMK, EEAHEFK, EEAHEYK, EEAHEWKEEGNTTPK, and EELDARLEALK. In another embodiment, the N-terminal extension is selected from QPMYKR, GQPMYK, PGQPMY, KPGQPM, LKPGQP, QLKPGQ, LQLKPG, WLQLKP, HWLQLK, WHWLQL, AWHWLQ, EAWHWL, AEAWHW, and EAEAWH.

[0121] When the expressed polypeptide contains an N-terminal extension, the N-terminal extension is customarily removed by using a protease, a peptidase, or by chemical cleavage. Proteases such as trypsin, Acromobacter lyticus protease, and enterokinase can be used. The specific proteolytic enzyme chosen for cleavage generally depends on the polypeptide produced. Thus, a person skilled in the art will typically select the proteolytic enzyme based on the polypeptide sequence, especially the presence of any internal major or minor cleavage sites, and adapting the N-terminal extension to form a good cleavage site.

[0122] When used to cleave an expressed polypeptide having an N-terminal extension, the cleavage efficiency of a protease can be determined by a simple assay as follows: An appropriate aqueous solution of the polypeptide is incubated at a pH and temperature favorable for the protease, and samples are taken from the reaction mixture over time. The enzyme activity is inactivated as soon as a sample is taken. After collecting all samples covering the period of interest, the concentration of the corresponding polypeptide without the N-terminal extension is determined by, for example, HPLC analysis. Plotting the concentration of the cleaved polypeptide as a function of time will indicate the progress of the reaction. Comparing such reaction profiles for different N-terminal extensions of the polypeptide will allow ranking of the N-terminal extensions based on the ability of the protease to release the polypeptide without the N-terminal extension.

[0123] The nucleic acid construct encoding the polypeptide can suitably be of genomic, cDNA, or synthetic origin. Alterations in the amino acid sequence are accomplished via modification of the genetic code by well-known techniques.

[0124] The DNA sequence encoding the polypeptide is usually inserted into a recombinant vector, which can be any vector that can conveniently undergo recombinant DNA procedures, and the choice of vector will generally depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector can be a vector that, when introduced into a host cell, is integrated into the host cell genome and replicated along with the chromosome into which it is integrated.

[0125] The vector is preferably an expression vector in which the DNA sequence encoding the polypeptide is operably linked to other segments required for DNA transcription. The term "operably linked" means that these segments are arranged so that they function in concert for their intended purpose, e.g., transcription begins in the promoter and continues through the DNA sequence encoding the polypeptide until it terminates within the terminator.

[0126] Thus, an expression vector for expressing a polypeptide will contain a promoter capable of initiating and directing transcription of the cloned gene or cDNA. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein homologous or heterologous to the host cell.

[0127] In addition, the expression vector for expressing a polypeptide will also contain a terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention.

[0128] The purpose of expressing the polypeptide is to be directed to the secretory pathway for extracellular expression into the growth medium. For example, useful signal peptides for expression in yeast host cells are obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase as propeptides in the leader sequence. Other examples of useful propeptides (signal peptides) are the aspartyl protease 3 (Yps1) signal peptide (Egel-Mitani et al. (1990) YEAST 6:127-137 and US 5,726,038), the α-factor signal of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathern et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and US 4,870,008), the signal peptide of mouse salivary amylase (O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), the modified carboxypeptidase signal peptide (L.A. Valls et al., Cell 48, 1987, pp. 887-897), and the yeast BAR1 signal peptide (WO 87 / 02670).

[0129] Procedures for ligating the DNA sequence encoding the polypeptide, the promoter, the terminator, and the secretory signal sequence, respectively, and for inserting them into a suitable vector containing the information required for replication are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

[0130] Many yeast cells contain an endogenous plasmid called the 2-micron plasmid, which contains various elements that ensure its stable maintenance in yeast cells (Guerineau et al., 1971, Biochem. Biophys. Res. Comm. 42(3):550-557). All or part of this endogenous 2-micron plasmid can be used together with a recombinant gene as a method for ensuring the stable maintenance of the sequences required for recombinant expression (Beggs J.D., 1978, Transformation of yeast by a replicating hybrid plasmid, Nature, 275:104-109). The inventors have found that when the endogenous 2-micron plasmid is present in the cell, the expression plasmid for recombinant expression only needs to contain an origin of replication and the STB region. Other factors present on the endogenous 2-micron plasmid can act in trans. The origin of replication and the STB region only constitute a small part of the endogenous 2μ plasmid.

[0131] In one aspect, the present invention provides an expression plasmid that only contains the origin of replication and the STB region from the 2-micron plasmid. Thus, this minimal expression plasmid does not contain any of the FLP region, repeat 1 region, REP1 region, D-protein, repeat 2 region, and REP2 region. In one embodiment, the plasmid contains an expression cassette, an E. coli part (including the AmpR gene), and a Schizosaccharomyces pombe (S. pombe) sequence encoding triose phosphate isomerase as described in Russell, P.R. (1985, Transcription of the triose-phosphate gene of Schizosaccharomyces pombe initiates from a startpoint different from that in Saccharomyces cerevisiae, Gene, 40:125-130). In another embodiment, the minimal plasmid does not contain the AmpR or other antibiotic resistance genes. Such antibiotic resistance genes are useful during cloning work in, for example, E. coli, but are preferably eliminated from plasmids used for industrial-scale recombinant protein expression. The antibiotic resistance gene can be made non-functional or removed from the host cell by well-known procedures, see, for example, WO 00 / 04172. The minimal expression plasmid can be used for the expression of polypeptides in yeast.

[0132] The vector of the present invention preferably contains one or more selectable markers, which allow for the easy selection of transformed cells. A selectable marker is a gene whose product provides insecticide or virus resistance, heavy metal resistance, complementation of auxotrophy, etc. Examples of bacterial selectable markers are the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers conferring antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in auxotrophic yeast cells include ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. A preferred selectable marker for yeast is the Schizosaccharomyces pombe TPI gene (Russell (1985) Gene 40:125-130).

[0133] In the vector, the polynucleotide sequence is operably linked to a suitable promoter sequence. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. Examples of promoters useful in yeast host cells are the Saccharomyces cerevisiae MFα1, TPI1, ADH2, TDH3 or PGK1 promoters.

[0134] The polynucleotide construct of the present invention will generally also be operably linked to a suitable terminator. In yeast, examples of suitable terminators are the TPI1 terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434), but any endogenous yeast terminator can also be used.

[0135] Procedures for ligating the polynucleotide sequences, promoters and terminators of the present invention, respectively, and for inserting them into a suitable yeast vector containing the information required for yeast replication are well known to those skilled in the art. It should be understood that the vector can be constructed as follows: First, a DNA construct containing the complete DNA sequence encoding the polypeptide to be expressed is prepared, and then this fragment is inserted into a suitable expression vector, or DNA fragments containing the genetic information of individual elements (such as the mating factor α variant of the present invention, optionally containing the polypeptide to be expressed with an N-terminal extension) are inserted sequentially, and then these elements are assembled by ligation, seamless cloning methods or by direct cloning via homologous recombination in yeast cells.

[0136] The present invention also relates to recombinant host cells comprising a polynucleotide sequence encoding the mating factor α variant of the present invention and the polypeptide to be expressed. A vector containing such a polynucleotide sequence is introduced into the host cell such that the vector remains as a chromosomal component or a self-replicating extrachromosomal vector.

[0137] As used herein, "host cell" is intended to mean a microorganism used for expressing a polypeptide of interest. A host cell includes any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0138] Suitable host cells for the present invention are yeast cells. As used herein, "yeast" includes basidiosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Basidiosporogenous yeasts are divided into the Spermophthoraceae and the Saccharomycetaceae. The latter includes four subfamilies: the Schizosaccharomycoideae (e.g., Schizosaccharomyces), the Nadsonioideae, the Lipomycoideae, and the Saccharomycoideae (e.g., Pichia, Kluyveromyces, and Saccharomyces). Basidiosporogenous yeasts include Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families: the Sporobolomycetaceae (e.g., Sorobolomyces and Bullera) and the Cryptococcaceae (e.g., Candida). Since the classification of yeasts may change in the future, for the purposes of the present invention, yeasts should be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).The biology of yeast and the manipulation of yeast genetics are well known in the art (see, for example, Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B. J. and Stopani, A. O. M., eds., 2nd ed., 1987; The Yeasts, Rose, A. H. and Harrison, J. S., eds., 2nd ed., 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).

[0139] The yeast host cells used in the methods of the invention can be any suitable yeast organism which, once cultured, produces large amounts of the polypeptide to be expressed.

[0140] Examples of suitable yeast organisms are strains of cells of species selected from the genera Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, and Yarrowia. In one embodiment, the yeast host cell is selected from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Schizosaccharomyces pombe, Sacchoromyces uvarum, Pichia kluyveri, Yarrowia lipolytica, Candida utilis, Candida cacaoi, and Geotrichum fermentans. Other useful yeast host cells are Kluyveromyces lactis, Kluyveromyces fragilis, Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, Schizosaccharomyces pombe, Ustilgo maydis, Candida maltose, Pichia guillermondii, and Pichia methanoliol (see Gleeson et al., J.Gen.Microbiol.132 , 1986, pp. 3459-3465; US 4,882,279 and US 4,879,231). For example, transformation of yeast cells can be achieved by protoplast formation and subsequent transformation in a manner known per se.

[0141] The host cell for expressing the polypeptide is preferably a cell that does not contain any functional antibiotic resistance gene. Although such antibiotic resistance genes are useful during the initial cloning steps in, for example, Escherichia coli, the antibiotic resistance genes can be made non-functional or removed from the host cell by known procedures, see, for example, WO 00 / 04172.

[0142] As used herein, "culture medium" is intended to denote a liquid solution for culturing host cells, i.e., for supporting the growth and product formation of yeast. Suitable yeast culture media are, for example, YPD or as described in WO2008 / 037735. The culture medium contains at least one carbon source, one or several nitrogen sources, essential salts including potassium salts, sodium salts, magnesium salts, phosphates and sulfates, trace metals, water-soluble vitamins, and processing aids including, but not limited to, antifoaming agents, protease inhibitors, stabilizers, ligands and inducers. Typical carbon sources are, for example, monosaccharides or disaccharides. Typical nitrogen sources are, for example, ammonia, urea, amino acids, yeast extract, corn steep liquor and fully or partially hydrolyzed proteins. Typical trace metals are, for example, Fe, Zn, Mn, Cu, Mo and H 3 BO 3 . Typical water-soluble vitamins are, for example, biotin, pantothenate, niacin, thiamine, p-aminobenzoic acid, choline, pyridoxol, folic acid, riboflavin and ascorbic acid.

[0143] As used herein, "fermentation" is intended to denote a sterile process for multiplying microorganisms immersed in a liquid culture medium. Fermentation is preferably carried out in a sterile stirred tank having a supply line for adding compressed sterile gases including, but not limited to, air, oxygen and ammonia. The fermenter may contain sensors and devices for monitoring pH, temperature, pressure, stirring rate, dissolved oxygen level, liquid content, foam level, feed rate and acid and alkali addition rates. In addition, the fermenter may be equipped with optics for monitoring the level of cell density, the concentration of metabolites and products (irrespective of their physical and chemical form).

[0144] The desired product produced during the fermentation process exists as a soluble extracellular substance, or as an intracellular substance in the form of a soluble substance or an insoluble substance including aggregated substances. The desired product preferably exists as a soluble extracellular substance. The fermentation process is usually carried out in a tank having a working volume of 100 mL to 200,000 L. The fermentation process can be operated as a batch process, a fed-batch process, a repeated fed-batch process or a continuous process.

[0145] The secreted polypeptides - most of which will be present in the culture medium in a properly processed form - can be recovered from the culture medium by conventional procedures, which include separating yeast cells from the culture medium by centrifugation, filtration, or capturing the polypeptides by an ion exchange matrix or by a reverse phase adsorption matrix, precipitating the protein components of the supernatant or filtrate by relying on salts such as ammonium sulfate, and then purifying by various chromatographic procedures such as ion exchange chromatography, affinity chromatography, etc.

[0146] The novel mating factor α variant of the present invention also facilitates the reduction of O-glycosylation of polypeptides during expression in yeast. Thus, the mating factor α variant of the present invention can be used in an improved method for preparing polypeptides such as GLP-1 peptides in yeast. Expressing a polypeptide in yeast cells with reduced O-glycosylation capacity can maintain an increased yield of the precursor, while at the same time even further reducing the proportion of the polypeptide that is O-glycosylated during expression.

[0147] Protein O-mannosyltransferase (PMT) initiates the assembly of O-mannosyl glycans, which are essential protein modifications in fungi. PMT is conserved in fungi, and the PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. By transferring mannose residues from dolichyl phosphate-D-mannose, the protein O-mannosyltransferases Pmt1p and Pmt2p catalyze the O-glycosylation of serine and threonine residues in proteins in the endoplasmic reticulum of yeast (Gentzsch et al., FEBS Lett 1995, 18, pp128-130). In Saccharomyces cerevisiae and in many other yeasts, the PMT family is highly redundant, and only the simultaneous deletion of PMT1 / PMT2 and PMT4 subfamily members is lethal (Girrbach and Strahl, J. Biol. Chem. 2003, 278, pp12554-62). US 5,714,377 describes that yeast cells with reduced O-glycosylation capacity due to PMT1 / PMT2 modification are still viable and exhibit good growth characteristics under industrial fermentation conditions.

[0148] Non-limiting embodiments

[0149] The present invention is further described by the following non-limiting embodiments:

[0150] 1. A method for recombinant expression of a polypeptide in yeast, which comprises culturing a yeast strain comprising a DNA sequence encoding a processing and secretion signal upstream of the polypeptide, wherein the processing and secretion signal comprises a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42 to comprise an amino acid sequence of general formula (I):

[0151] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0152] Wherein

[0153] X 38 is F, L, I or V;

[0154] X 39 is L, I, V or M;

[0155] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0156] X 41 is S, Y, F, W, L, I, V or M;

[0157] X 42 is Y, W, L, I, V, M or S;

[0158] Condition is X 38 -X 42 is not VIGYS.

[0159] 2. The method according to embodiment 1, wherein

[0160] X 38 is F, L or V;

[0161] X 39 is L, I, V or M;

[0162] X 40 is G or R;

[0163] X 41 is S, Y, L, I, V or M, and

[0164] X 42 is Y, W, L, V or M.

[0165] 3. The method according to claim 1, wherein

[0166] X 38 is I or V;

[0167] X 39 is L, I, V or M;

[0168] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0169] X 41 is Y, F or W, and

[0170] X 42 is L or I.

[0171] 4. The method according to any one of embodiments 1 - 3, wherein

[0172] X 38 is V;

[0173] X 39 is L, I, V or M;

[0174] X 40 is G or R;

[0175] X 41 is Y, and

[0176] X 42 is L.

[0177] 5. The method according to any one of embodiments 1 - 4, wherein X 38 -X 39 -X 40 -X 41 -X 42 four of the amino acid residues in are the same as the corresponding amino acid residues in VIGYL or VIGYS.

[0178] 6. The method according to any one of embodiments 1 - 5, wherein X 38 -X 39 -X 40 -X 41 -X 42 at least three of the amino acid residues in are the same as the corresponding amino acid residues in VIGYL or VIGYS.

[0179] 7. The method according to any one of embodiments 1 - 6, wherein X 41 is Y.

[0180] 8. The method according to any one of embodiments 1 - 6, wherein X 41 is L.

[0181] 9. The method according to any one of embodiments 1 - 8, wherein X 42 is L.

[0182] 10. The method according to any one of embodiments 1 and 5 - 8, wherein X 42 is S.

[0183] 11. The method according to any one of embodiments 1 and 5 - 8, wherein X 42 is Y, W, L, I, V or M.

[0184] 12. The method according to any one of embodiments 1 - 11, wherein X 40 is R.

[0185] 13. The method according to embodiment 12, wherein X 38 is V, X 39 is I, X 41is Y, and X 42 is L.

[0186] 14. The method according to embodiment 12, wherein X 38 is V, X 39 is I, X 41 is Y, and X 42 is S.

[0187] 15. The method according to any one of embodiments 1 and 12, wherein formula (I) is VI-X 40 -YL or VI-X 40 -YS.

[0188] 16. The method according to embodiment 15, wherein X 40 is A, Y, F, W, R, K, L, I, V or M.

[0189] 17. The method according to any one of embodiments 1-16, wherein compared with the mating factor α propeptide (amino acid residues 20-85) shown in SEQ ID NO: 2, the mating factor α propeptide variant has less than 10 amino acid residue changes outside the X 38 -X 42 sequence.

[0190] 18. The method according to any one of embodiments 1-17, wherein compared with the mating factor α propeptide (amino acid residues 20-85) shown in SEQ ID NO: 2, the mating factor α propeptide variant has less than 5 amino acid residue changes outside the X 38 -X 42 sequence.

[0191] 19. The method according to any one of embodiments 1-18, wherein compared with the mating factor α propeptide (amino acid residues 20-85) shown in SEQ ID NO: 2, the mating factor α propeptide variant has less than 2 amino acid residue changes outside the X 38 -X 42 sequence.

[0192] 20. The method according to any one of embodiments 1-19, wherein the mating factor α propeptide variant is part of a mating factor α prepropeptide that comprises a propeptide as an N-terminal portion fused to the mating factor α propeptide variant as a C-terminal portion.

[0193] 21. The method according to embodiment 20, wherein the propeptide is from the yeast aspartic protease 3 (YAP3) signal peptide, the α-factor signal from the MFα1 gene of Saccharomyces cerevisiae or a variant thereof.

[0194] 22. A method according to any one of embodiments 1-21, wherein the yeast carries at least one genetic modification that reduces its O-glycosylation ability.

[0195] 23. A method according to embodiment 22, wherein the yeast carries at least one genetic modification within the gene of PMT1 or PMT2 that reduces its O-glycosylation ability.

[0196] 24. A method according to any one of embodiments 22-23, wherein the yeast carries at least one genetic modification that reduces its ability to O-glycosylate the polypeptide GLP-1(7-37)[K34R] expressed together with an α-leader sequence by protein O-mannosyltransferase 1 (PMT1), compared to a yeast carrying the corresponding unmodified gene.

[0197] 25. A method according to any one of embodiments 22-24, wherein the yeast carries at least one genetic modification that reduces its ability to O-glycosylate the polypeptide GLP-1(7-37)[K34R] expressed together with an α-leader sequence by protein O-mannosyltransferase 2 (PMT2), compared to a yeast carrying the corresponding unmodified gene.

[0198] 26. A method according to any one of embodiments 22-25, wherein the O-glycosylation ability is reduced by at least 2-fold.

[0199] 27. A method according to any one of embodiments 22-26, wherein the O-glycosylation ability is reduced by at least 4-fold.

[0200] 28. A method according to any one of embodiments 22-27, wherein the at least one genetic modification is located in the coding region of PMT1 or PMT2.

[0201] 29. A method according to any one of embodiments 22-27, wherein the at least one genetic modification is located in a region responsible for or involved in the expression and / or transcriptional regulation of PMT1 or PMT2.

[0202] 30. A method according to any one of embodiments 22-27, wherein the PMT1 gene in the yeast is deleted.

[0203] 31. A method according to any one of embodiments 22-27, wherein both the PMT1 and PMT2 genes in the yeast are deleted.

[0204] 32. A method according to any one of embodiments 1-31, wherein the polypeptide comprises a GLP-1 peptide.

[0205] 33. The method according to embodiment 32, wherein the polypeptide comprises GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R].

[0206] 34. The method according to embodiment 33, wherein the polypeptide is GLP-1(7-37)[K34R], GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K] (SEQ ID NO:44).

[0207] 35. The method according to any one of embodiments 32-34, wherein the polypeptide has an N-terminal extension.

[0208] 36. The method according to embodiment 35, wherein the N-terminal extension is selected from EEK, EEAEK, HK, EEAHK, E(EA)2HK, E(EA)3HK, EEGHK, EHPK, EEGEPK, EEAHELK, EEAHEVK, EEAHEMK, EEAHEFK, EEAHEYK, EEAHEWKEEGNTTPK, and EELDARLEALK.

[0209] 37. The method according to embodiment 35, wherein the N-terminal extension is selected from DV, DVKPGQPLA, DVKPGQPEY, DVKPGEPLY, DVKPGQPLY, DVKPGQPLE, DVKPGQPMY, and DVKPGQPMYDDDDK.

[0210] 38. The method according to embodiment 35, wherein the N-terminal extension is selected from QPMYKR, GQPMYK, PGQPMY, KPGQPM, LKPGQP, QLKPGQ, LQLKPG, WLQLKP, HWLQLK, WHWLQL, AWHWLQ, EAWHWL, AEAWHW, and EAEAWH.

[0211] 39. A mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42, thereby comprising an amino acid sequence of general formula (I):

[0212] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0213] wherein

[0214] X 38 is F, L, I or V;

[0215] X 39 is L, I, V or M;

[0216] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0217] X 41 is S, Y, F, W, L, I, V or M;

[0218] X 42 is Y, W, L, I, V, M or S;

[0219] Provided that X 38 -X 42 is not VIGYS.

[0220] 40. The mating factor α propeptide variant according to embodiment 39, wherein X 38 -X 42 is not VIGYS, VIDYS, VATYL, VIGYR or AIGYL.

[0221] 41. A GLP-1 precursor, which is a fusion polypeptide comprising:

[0222] - a propeptide,

[0223] - a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42, thereby comprising an amino acid sequence of general formula (I):

[0224] X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I)

[0225] wherein

[0226] X 38 is F, L, I or V;

[0227] X 39 is L, I, V or M;

[0228] X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;

[0230] X 41 is S, Y, F, W, L, I, V or M;

[0231] X 42 is Y, W, L, I, V, M or S;

[0232] The condition is X 38 -X 42 is not VIGYS,

[0233] -an optional extension peptide, and

[0234] -a GLP-1 peptide.

[0235] 42. The GLP-1 precursor according to embodiment 41, wherein the peptides comprised by the GLP-1 precursor are fused in the order in which they are listed, i.e., the propeptide - mating factor alpha propeptide variant, an optional extension peptide, and a GLP-1 peptide.

[0236] 43. A DNA sequence encoding the polypeptide according to any one of embodiments 39 - 42.

[0237] 44. An expression vector comprising the DNA sequence according to embodiment 43.

[0238] 45. The expression vector according to embodiment 44, wherein the DNA sequence encoding the polypeptide to be expressed is operably linked to an upstream promoter and a downstream terminator.

[0239] 46. A host cell comprising the expression vector according to any one of embodiments 44 - 45.

[0240] 47. The host cell according to embodiment 46, which is selected from species of the genus Saccharomyces, species of the genus Pichia, species of the genus Hansenula, species of Arxula, species of the genus Kluyveromyces, species of the genus Yarrowia, and species of the genus Schizosaccharomyces.

[0241] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference in their entirety as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and subheadings used herein are for convenience only and should not be construed as limiting the invention in any way. Unless otherwise stated, any and all examples or the use of exemplary language provided herein (e.g., "such as") are only intended to better illustrate the invention and do not limit the scope of the invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention. The citation and incorporation of patent documents herein are made for convenience only and do not reflect any opinion as to the validity, patentability, and / or enforceability of such patent documents. As permitted by applicable law, the invention includes all modifications and equivalents of the subject matter recited in the appended claims.

[0242] Example

[0243] Examples 1 - 45

[0244] We constructed a plasmid containing the TDH3 promoter, a gene encoding the MFalpha prepropeptide, a gene encoding an N - terminal extended GLP - 1 (DVKPGQPMYDDDDK - GLP - 1(7 - 37)[K34R]) (SEQ ID NO:45), a minimal 2 - micron region for maintenance in yeast, and a selectable marker, namely the TPI gene from Schizosaccharomyces pombe (see Figure 1 ).

[0245] In this plasmid background, we introduced various single mutations in the region of positions 38 - 42 in the region encoding the MFalpha - prepropeptide region, X 38 -X 42 =VIGYL sequence (see Figure 2 ). The experiment using the wild - type VIGYL sequence was Example 1, see Tables 1 to 5. Other reference experiments were Examples 5 - 6, 10 - 11, 28 - 29, 37 - 38, and 44 - 45.

[0246] These plasmids were introduced into a Saccharomyces cerevisiae strain lacking the TPI1 gene to allow selection of transformants carrying the plasmid on a medium containing glucose as the sole carbon source. Thereafter, the transformants were cultured in 5 ml of the relevant medium in a shake flask for 3 days, and the concentration of the secreted GLP - 1 peptide and the degree of O - glycosylation of this peptide in the culture supernatant were analyzed by LCMS. The yields and degrees of O - glycosylation of the wild - type and single - amino - acid residue mutants (including a pair of reference examples at each position) of the X 38 -X 42 sequence are shown in Tables 1 to 5.

[0247] Table 1. Data on the expression of N - terminal extended GLP - 1 under the control of a mating factor α prepropeptide mutant with a single - amino - acid mutation at position 38 (X 38 ). The concentrations of N - terminal extended GLP - 1 and O - glycosylated (O - glyco) impurities were normalized against data from the same expression under the control of a mating factor α prepropeptide with wild - type Val as X 38 .

[0248]

[0249] Table 2. At position 39 (X 39Data on the expression of N-terminally extended GLP-1 under the control of a mating factor α propeptide mutant with a single amino acid mutation. The concentrations of N-terminally extended GLP-1 and O-glycosylated (O-glyco) impurities were normalized against data from the same expression under the control of a mating factor α propeptide with wild-type Ile as X 39 from the same expression.

[0250]

[0251] Table 3. Data on the expression of N-terminally extended GLP-1 under the control of a mating factor α propeptide mutant with a single amino acid mutation at position 40 (X 40 ). The concentrations of N-terminally extended GLP-1 and O-glycosylated (O-glyco) impurities were normalized against data from the same expression under the control of a mating factor α propeptide with wild-type Gly as X 40 from the same expression.

[0252]

[0253] Table 4. Data on the expression of N-terminally extended GLP-1 under the control of a mating factor α propeptide mutant with a single amino acid mutation at position 41 (X 41 ). The concentrations of N-terminally extended GLP-1 and O-glycosylated (O-glyco) impurities were normalized against data from the same expression under the control of a mating factor α propeptide with wild-type Tyr as X 41 from the same expression.

[0254]

[0255]

[0256] Table 5. Data on the expression of N-terminally extended GLP-1 under the control of a mating factor α propeptide mutant with a single amino acid mutation at position 42 (X 42 ). The concentrations of N-terminally extended GLP-1 and O-glycosylated (O-glyco) impurities were normalized against data from the same expression under the control of a mating factor α propeptide with wild-type Leu as X 42 from the same expression.

[0257]

[0258] Example 46.

[0259] A plasmid similar to the plasmids in Examples 1 - 45, which contains the TDH3 promoter, a gene encoding the MFalpha prepropeptide, a gene encoding the N - terminal extended GLP - 1 (DVKPGQPMYDDDDK - GLP - 1(7 - 37)[K34R]), a minimal 2 - micron region for maintenance in yeast, and a selectable marker( Figure 1 ), and this plasmid was transformed into two different strain backgrounds - one containing the PMT1 gene (PMT+), and the other lacking the PMT1 gene (PMT -). The PMT1 gene encodes the protein mannosyltransferase involved in O - glycosylation.

[0260] The MFalpha prepropeptide is wild - type (40G) or mutant (G40R). The strains were cultured under the same conditions in continuous culture, dilution rate = 0.1, and pH 5.8. Samples were analyzed by HPLC to determine the concentration of GLP - 1 precursor in the spent medium, and by LCMS analysis to determine the proportion of O - glycosylated GLP - 1 precursor.

[0261] The results demonstrated that the effects were indeed additive.

[0262] The G40R mutation reduced O - glycosylation by more than 80%. The deletion of PMT1 further reduced O - glycosylation by more than 80%, resulting in an O - glycosylation level close to the detection limit in this case. In addition, in both the PMT+ strain and the pmt1 - deleted strain, the G40R mutation led to a remarkable increase in the yield of N - terminal extended GLP - 1 compared to the 40G wild - type form (see Table 6).

[0263] These results indicate that the combination of a beneficial mutation such as G40R in the MFalpha prepropeptide and a PMT1 - deleted host strain results in an additive reduction in O - glycosylation.

[0264] Table 6. Normalized maximum concentration (yield) of the peptide DVKPGQPMYDDDDK - GLP - 1(7 - 37)[K34R] in continuous culture when using the 40R - mutant MFalpha prepropeptide compared to the corresponding wild - type 40G.

[0265]

[0266] Example 47.

[0267] To investigate the role of one of said mutations in the expression process of other GLP-1 peptides, we constructed plasmids containing the TDH3 promoter, a gene encoding the wild-type (40G) or 40R mutant form of the MFalpha prepropeptide, a gene encoding an N-terminally extended GLP-1 (DVKPGQPMYDDDDK-GLP-1(9-37)[K34R] or DVKPGQPMYDDDDK-GLP-1(9-37)[K34R,G37K]), a minimal 2-micron region for maintenance in yeast, and a selectable marker, namely the TPI gene from Schizosaccharomyces pombe (see Figure 1 ).

[0268] These plasmids were introduced into a Saccharomyces cerevisiae strain lacking the TPI1 gene to allow selection of transformants carrying the plasmid on a medium containing glucose as the sole carbon source. The transformants were then cultured for 3 days in 5 ml of the relevant medium in a shake flask, and the concentration of the secreted GLP-1 peptide and the degree of O-glycosylation of the peptide in the culture supernatant were analyzed by LCMS. Table 7 shows the results for the wild-type (wt) and 40R mutations upon expression, including the yield of the GLP-1 peptide and the degree of O-glycosylation of the GLP-1 peptide.

[0269] Table 7. Data on the expression of a specifically N-terminally extended GLP-1 peptide under the control of a mating factor alpha propeptide mutant with or without a single amino acid substitution to Arg at position 40 (X 40 ). The N-terminally extended GLP-1 peptide (yield) and the concentration of O-glycosylated impurities were normalized against data from the expression of the same GLP-1 peptide under the control of a mating factor alpha propeptide with wild-type Gly as X 40 .

[0270]

[0271] BDL: Below detection limit

[0272] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, variations, and equivalents will now occur to those of ordinary skill in the art. Accordingly, it is to be understood that the appended claims are intended to cover all such modifications and variations that fall within the true spirit of the present invention.

Claims

1. A method for recombinant expression in yeast of a polypeptide comprising a GLP-1 peptide, which comprises culturing a yeast strain comprising a DNA sequence encoding a processing and secretion signal upstream of the polypeptide, wherein the processing and secretion signal comprises a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42 such that it comprises an amino acid sequence of general formula (I): X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I) wherein X 38 is F, L, I or V; X 39 is L, I, V or M; X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; X 41 is S, Y, F, W, L, I, V or M; X 42 is Y, W, L, I, V, M, or S; The condition is X 38 -X 42 It is not VIGYS.

2. The method according to claim 1, wherein X 38 is F, L, or V; X 39 is L, I, V, or M; X 40 is G or R; X 41 is S, Y, L, I, V or M, and X 42 is Y, W, L, V, or M.

3. The method according to claim 1, wherein X 38 is I or V; X 39 is L, I, V or M; X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; X 41 is Y, F, or W, and X 42 is L or I.

4. The method according to any one of claims 1-3, wherein X 38 is V; X 39 is L, I, V, or M; X 40 is G or R; X 41 is Y, and X 42 is L.

5. The method according to any one of claims 1-4, wherein X 40 is R.

6. The method according to any one of claims 1-5, wherein the mating factor α propeptide variant has less than 10 amino acid residue changes outside the sequence of -X 38 -X 42 compared to the mating factor α propeptide (amino acid residues 20-85) shown in SEQ ID NO:

2. 38 -X 42 ​ 7. The method according to any one of claims 1-6, wherein the yeast carries at least one genetic modification that reduces its O-glycosylation ability.

8. The method according to claim 7, wherein the PMT1 gene in the yeast is deleted.

9. The method according to any one of claims 1-8, wherein the polypeptide comprises GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K].

10. The method according to any one of claims 1-9, wherein the polypeptide consists of GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R].

11. The method according to any one of claims 9-10, wherein the polypeptide has an N-terminal extension.

12. A mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42 such that it comprises an amino acid sequence of general formula (I): X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I) wherein X 38 is F, L, I or V; X 39 is L, I, V, or M; X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; X 41 is S, Y, F, W, L, I, V or M; X 42 is Y, W, L, I, V, M, or S; Condition is X 38 -X 42 Not VIGYS, VI D YS, V AT Y L , VIGY R Or A IGY L .

13. A GLP-1 precursor, which is a fusion polypeptide comprising: - a propeptide, - a mating factor α propeptide variant having at least one substitution in the VIGYL sequence at positions 38-42 such that it comprises an amino acid sequence of general formula (I): X 38 -X 39 -X 40 -X 41 -X 42 (SEQ ID NO:1)(I) wherein X 38 is F, L, I or V; X 39 is L, I, V or M; X 40 is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; X 41 is S, Y, F, W, L, I, V or M; X 42 is Y, W, L, I, V, M or S; Condition is X 38 -X 42 not VIGYS, - an optional extension peptide, and - a GLP-1 peptide.

14. An expression vector comprising a DNA sequence encoding the polypeptide according to any one of claims 12-13.

15. A host cell comprising the expression vector according to claim 14.

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