Method To Engineer Mammanlian-type Carbohydrate Structures

Abstract

The present invention relates to host cells having modified lipid-linked oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells have a GlcNAcMan.sub.3GlcNAc.sub.2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No. 10/500,240, filed Mar. 23, 2005, now pending, which is a 371 National Stage Application No. PCT/US02/41510, filed Dec. 24, 2002, which claims the benefit of U.S. provisional application Ser. No. 60/344,169, Dec. 27, 2001, which is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name "GF0022PDA_SEQLIST.txt", creation date of Aug. 6, 2014, and a size of 91 KB. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention generally relates to modifying the glycosylation structures of recombinant proteins expressed in fungi or other lower eukaryotes, to more closely resemble the glycosylation of proteins of higher mammals, in particular humans.

BACKGROUND OF THE INVENTION

[0004] After DNA is transcribed and translated into a protein, further post translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of one and the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins, and if so only in a very unspecific manner (Moens, 1997). Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars, whereas insect cells such as Sf9 cells glycosylate proteins in yet another way. See for example (Bretthauer, 1999; Martinet, 1998; Weikert, 1999; Malissard, 2000; Jarvis, 1998; and Takeuchi, 1997).

[0005] Synthesis of a mammalian-type oligosaccharide structure consists of a series of reactions in the course of which sugar-residues are added and removed while the protein moves along the secretory pathway in the host organism. The enzymes which reside along the glycosylation pathway of the host organism or cell determine what the resulting glycosylation patterns of secreted proteins. Unfortunately, the resulting glycosylation pattern of proteins expressed in lower eukaryotic host cells differs substantially from the glycosylation found in higher eukaryotes such as humans and other mammals (Bretthauer, 1999). Moreover, the vastly different glycosylation pattern has, in some cases, been shown to increase the immunogenicity of these proteins in humans and reduce their half-life (Takeuchi, 1997). It would be desirable to produce human-like glycoproteins in non-human host cells, especially lower eukaryotic cells.

[0006] The early steps of human glycosylation can be divided into at least two different phases: (i) lipid-linked Glc.sub.3Man.sub.9GlcNAc.sub.2 oligosaccharides are assembled by a sequential set of reactions at the membrane of the endoplasmic reticulum (ER) and (ii) the transfer of this oligosaccharide from the lipid anchor dolichyl pyrophosphate onto de novo synthesized protein. The site of the specific transfer is defined by an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr (see FIG. 1), where Xaa can be any amino acid except proline (Gavel, 1990). Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the early Golgi apparatus, where additional mannose residues are removed by Golgi specific alpha (.alpha.)-1,2-mannosidases. Processing continues as the protein proceeds through the Golgi. In the medial Golgi, a number of modifying enzymes, including N-acetylglucosaminyltransferases (GnT I, GnT II, GnT III, GnT IV GnT V GnT VI), mannosidase II and fucosyltransferases, add and remove specific sugar residues (see, e.g., FIGS. 2 and 3). Finally, in the trans-Golgi, galactosyltranferases and sialyltransferases produce a glycoprotein structure that is released from the Golgi. It is this structure, characterized by bi-, tri- and tetra-antennary structures, containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid, that gives glycoproteins their human characteristics.

[0007] In nearly all eukaryotes, glycoproteins are derived from the common core oligosaccharide precursor Glc.sub.3Man.sub.9GlcNAc.sub.2-PP-Dol, where PP-Dol stands for dolichol-pyrophosphate (FIG. 1). Within the endoplasmic reticulum, synthesis and processing of dolichol pyrophosphate bound oligosaccharides are identical between all known eukaryotes. However, further processing of the core oligosaccharide by yeast, once it has been transferred to a peptide leaving the ER and entering the Golgi, differs significantly from humans as it moves along the secretory pathway and involves the addition of several mannose sugars.

[0008] In yeast, these steps are catalyzed by Golgi residing mannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentially add mannose sugars to the core oligosaccharide. The resulting structure is undesirable for the production of humanoid proteins and it is thus desirable to reduce or eliminate mannosyltransferase activity. Mutants of S. cerevisiae, deficient in mannosyltransferase activity (for example och1 or mnn9 mutants) have been shown to be non-lethal and display a reduced mannose content in the oligosacharide of yeast glycoproteins. Other oligosacharide processing enzymes, such as mannosylphophate transferase may also have to be eliminated depending on the host's particular endogenous glycosylation pattern.

Lipid-Linked Oligosaccharide Precursors

[0009] Of particular interest for this invention are the early steps of N-glycosylation (FIGS. 1 and 2). The study of alg (asparagine-linked glycosylation) mutants defective in the biosynthesis of the Glc.sub.3Man.sub.9GlcNAc.sub.2-PP-Dol has helped to elucidate the initial steps of N-glycosylation.

[0010] The ALG3 gene of S. cerevisiae has been successfully cloned and knocked out by deletion (Aebi, 1996). ALG3 has been shown to encode the enzyme Dol-P-Man:Man.sub.5GlcNAc.sub.2-PP-Dol Mannosyltransferase, which is involved in the first Dol-P-Man dependent mannosylation step from Man.sub.5GlcNAc.sub.2-PP-Dol to Man.sub.6GlcNAc.sub.2-PP-Dol at the luminal side of the ER (Sharma, 2001) (FIGS. 1 and 2). S. cerevisiae cells harboring a leaky alg3-1 mutation accumulate Man.sub.5GlcNAc.sub.2-PP-Dol (structure I) (Huffaker, 1983).

##STR00001##

Man.sub.5GlcNAc.sub.2 (Structure I) and Man.sub.8GlcNAc.sub.2 accumulate in total cell mannoprotein of an och1 mnn1 alg3 mutant(Nakanishi-Shindo, 1993). This S. cerevisiae och1, mnn1, alg3 mutant was shown to be viable, but temperature-sensitive, and to lack .alpha.-1,6 polymannose outer chains.

[0011] In another study, secretory proteins expressed in a strain deleted for alg 3 (.DELTA.alg3 background) were studied for their resistance to Endo-.beta.-N-acetylglucosaminidase H (Endo H) (Aebi, 1996). Previous observations have indicated that only those oligosaccharides larger than Man.sub.5GlcNAc.sub.2 are susceptible to cleavage by Endo H (Hubbard, 1980). In the alg3-1 phenotype, some glycoforms were sensitive to Endo H cleavage, confirming its leakiness, whereas in the .DELTA.alg3 mutant all glycoforms appeared to be resistant and of the Man.sub.s-type (Aebi, 1996), suggesting a tight phenotype and transfer of Man.sub.5GlcNAc.sub.2 oligosaccharide structures onto the nascent polypeptide chain. No obvious phenotype was connected with the inactivation of the ALG3 gene (Aebi, 1996). Secreted exogluconase produced in a Saccharomyces cerevisiae alg3 mutant was found to contain between 35-44% underglycosylated and unglycosylated forms and only about 50% of the transferred oligosaccharides remained resistant to Endo H treatment (Cueva, 1996). Exoglucanase (Exg), an enzyme that contains two potential N-glycosylation sites at Asn.sub.165 and Asn.sub.325, was analyzed in more detail. For Exg molecules that received two oligosaccharides it was shown that the first N-glycosylation site (Asn.sub.165) was enriched in truncated residues, whereas the second (Asn.sub.325) was enriched in regular oligosaccharides. 35-44% of secreted exoglucanase was non- or underglycosylated and about 73-78% of all available N-glycosylation sites were occupied with either truncated or regular oligosaccharides (Cueva, 1996).

Transfer of Glucosylated Lipid-Linked Oligosaccharides

[0012] Evidence suggests that, in mammalian cells, only glucosylated lipid-linked oligosaccharides are transferred to nascent proteins (Turco, 1977), while in yeast alg5, alg6 and dpg1 mutants, nonglucosylated oligosaccharideds can be transferred (Ballou, 1986; Runge, 1984). In a Saccharomyces cerevisiae alg8 mutant, underglucosylated GlcMan.sub.9GlcNAc.sub.2 is transferred (Runge, 1986). Verostek and co-workers studied an alg3, sec18, gls1 mutant and proposed that glucosylation of a Man.sub.5GlcNAc.sub.2 structure (Structure I, above) is relatively slow in comparison to glucosylation of a lipid-linked Man.sub.5 structure. In addition, the transfer of this Man.sub.5GlcNAc.sub.2 structure to protein appears to be about 5-fold more efficient than the glucosylation to Glc.sub.3Man.sub.5GlcNAc.sub.2. The decreased rate of Man.sub.5GlcNAc.sub.2 glucosylation in combination with the comparatively faster rate of Man.sub.5 structure transfer onto nascent protein is believed to be the cause of the observed accumulation of nonglucosylated Man.sub.5 structures in alg3 mutant yeast (Verostek-a, 1993; Verostek-b, 1993).

[0013] Studies preceding the above work did not reveal any lipid-linked glucosylated oligosaccharides (Orlean, 1990; Huffaker, 1983) allowing the conclusion that glucosylated oligosaccharides are transferred at a much higher rate than their nonglucosylated counterparts and thus are much harder to isolate. Recent work has allowed the creation and study of yeast strains with un- and hypoglucosylated oligosaccharides and has further confirmed the importance of the addition of glucose to the antenna of lipid-linked oligosaccharides for substrate recognition by the oligosaccharyltransferase complex (Reiss, 1996; Stagljar, 1994; Burda, 1998). The decreased degree of glucosylation of the lipid-linked Man.sub.5-oligosaccharides in an alg3 mutant negatively impacts the kinetics of the transfer of lipid-linked oligosaccharides onto nascent protein and is believed to be the cause for the strong underglycosylation of secreted proteins in an alg3 knock-out strain (Aebi, 1996).

[0014] The assembly of the lipid-linked core oligosaccharide Man.sub.9GlcNAc.sub.2 occurs, as described above, at the membrane of the endoplasmatic reticulum. The additions of three glucose units to the .alpha.-1,3-antenna of the lipid-linked oligosaccharides are the final reactions in the oligosaccharide assembly. First an .alpha.-1,3 glucose residue is added followed by another .alpha.-1,3 glucose residue and a terminal .alpha.-1,2 glucose residue. Mutants accumulating dolichol-linked Man.sub.9GlcNAc.sub.2 have been shown to be defective in the ALG6 locus, and Alg6p has similarities to Alg8p, the .alpha.-1,3-glucosyltransferase catalyzing the addition of the second .alpha.-1,3-linked glucose (Reiss, 1996). Cells with a defective ALG8 locus accumulate dolichol-linked Glc.sub.1Man.sub.9GlcNAc.sub.2 (Runge, 1986; Stagljar, 1994). The ALG10 locus encodes the .alpha.-1,2 glucosyltransferase responsible for the addition of a single terminal glucose to Glc.sub.2Man.sub.9GlcNAc.sub.2-PP-Dol (Burda, 1998).

Sequential Processing of N-glycans by Localized Enzyme Activities

[0015] Sugar transferases and mannosidases line the inner (luminal) surface of the ER and Golgi apparatus and thereby provide a "catalytic" surface that allows for the sequential processing of glycoproteins as they proceed through the ER and Golgi network. In fact the multiple compartments of the cis, medial, and trans Golgi and the trans-Golgi Network (TGN), provide the different localities in which the ordered sequence of glycosylation reactions can take place. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or TGN, it is sequentially exposed to different glycosidases, mannosidases and glycosyltransferases such that a specific carbohydrate structure may synthesized. Much work has been dedicated to revealing the exact mechanism by which these enzymes are retained and anchored to their respective organelle. The evolving picture is complex but evidence suggests that, stem region, membrane spanning region and cytoplasmic tail individually or in concert direct enzymes to the membrane of individual organelles and thereby localize the associated catalytic domain to that locus.

[0016] In some cases these specific interactions were found to function across species. For example the membrane spanning domain of .alpha.2,6-ST from rats, an enzyme known to localize in the trans-Golgi of the animal, was shown to also localize a reporter gene (invertase) in the yeast Golgi (Schwientek, 1995). However, the very same membrane spanning domain as part of a full-length .alpha.2,6 ST was retained in the ER and not further transported to the Golgi of yeast (Krezdorn, 1994). A full length Gal-Tr from humans was not even synthesized in yeast, despite demonstrably high transcription levels. On the other hand the transmembrane region of human the same GalT fused to an invertase reporter was able to direct localization to the yeast Golgi, albeit it at low production levels. Schwientek and co-workers have shown that fusing 28 amino acids of a yeast mannosyltransferase (Mnt1), a region containing a cytoplamic tail, a transmembrane region and eight amino acids of the stem region, to the catalytic domain of human GalT are sufficient for Golgi localization of an active GalT. Other galactosyltransferases appear to rely on interactions with enzymes resident in particular organelles since after removal of their transmembrane region they are still able to localize properly. To date there exists no reliable way of predicting whether a particular heterologously expressed glycosyltransferase or mannosidase in a lower eukaryote will be (1), sufficiently translated (2), catalytically active or (3) located to the proper organelle within the secretory pathway. Since all three of these are necessary to effect glycosylation patterns in lower eukaryotes, a systematic scheme to achieve the desired catalytic function and proper retention of enzymes in the absence of predictive tools, which are currently not available, has been designed.

Production of Therapeutic Glycoproteins

[0017] A significant number of proteins isolated from humans or animals are post-translationally modified, with glycosylation being one of the most significant modifications. An estimated 70% of all therapeutic proteins are glycosylated and thus currently rely on a production system (i.e., host cell) that is able to glycosylate in a manner similar to humans. To date, most glycoproteins are made in a mammalian host system. Several studies have shown that glycosylation plays an important role in determining the (1) immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4) efficacy of therapeutic proteins. It is thus not surprising that substantial efforts by the pharmaceutical industry have been directed at developing processes to obtain glycoproteins that are as "humanoid" or "human-like" as possible. This may involve the genetic engineering of such mammalian cells to enhance the degree of sialylation (i.e., terminal addition of sialic acid) of proteins expressed by the cells, which is known to improve pharmacokinetic properties of such proteins. Alternatively one may improve the degree of sialylation by in vitro addition of such sugars using known glycosyltransferases and their respective nucleotide sugars (e.g., 2,3 sialyltransferase and CMP-Sialic acid).

[0018] Future research may reveal the biological and therapeutic significance of specific glycoforms, thereby rendering the ability to produce such specific glycoforms desirable. To date, efforts have concentrated on making proteins with fairly well characterized glycosylation patterns, and expressing a cDNA encoding such a protein in one of the following higher eukaryotic protein expression systems:

[0019] 1. Higher eukaryotes such as Chinese hamster ovary cells (CHO), mouse fibroblast cells and mouse myeloma cells (Werner, 1998);

[0020] 2. Transgenic animals such as goats, sheep, mice and others (Dente, 1988); (Cole, 1994); (McGarvey, 1995); (Bardor, 1999);

[0021] 3. Plants (Arabidopsis thaliana, tobacco etc.) (Staub, 2000); (McGarvey, 1995); (Bardor, 1999);

[0022] 4. Insect cells (Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc., in combination with recombinant baculoviruses such as Autographa californica multiple nuclear polyhedrosis virus which infects lepidopteran cells (Altmann, 1999).

[0023] While most higher eukaryotes carry out glycosylation reactions that are similar to those found in humans, recombinant human proteins expressed in the above mentioned host systems invariably differ from their "natural" human counterpart (Raju, 2000). Extensive development work has thus been directed at finding ways to improving the "human character" of proteins made in these expression systems. This includes the optimization of fermentation conditions and the genetic modification of protein expression hosts by introducing genes encoding enzymes involved in the formation of human like glycoforms (Werner, 1998); (Weikert, 1999); (Andersen, 1994); (Yang, 2000). Inherent problems associated with all mammalian expression systems have not been solved.

[0024] Fermentation processes based on mammalian cell culture (e.g., CHO, murine, or human cells), for example, tend to be very slow (fermentation times in excess of one week are not uncommon), often yield low product titers, require expensive nutrients and cofactors (e.g., bovine fetal serum), are limited by programmed cell death (apoptosis), and often do not enable expression of particular therapeutically valuable proteins. More importantly, mammalian cells are susceptible to viruses that have the potential to be human pathogens and stringent quality controls are required to assure product safety. This is of particular concern since many such processes require the addition of complex and temperature sensitive media components that are derived from animals (e.g., bovine calf serum), which may carry agents pathogenic to humans such as bovine spongiform encephalopathy (BSE) prions or viruses. Moreover, the production of therapeutic compounds is preferably carried out in a well-controlled sterile environment. An animal farm, no matter how cleanly kept, does not constitute such an environment, thus constituting an additional problem in the use of transgenic animals for manufacturing high volume therapeutic proteins.

[0025] Most, if not all, currently produced therapeutic glycoproteins are therefore expressed in mammalian cells and much effort has been directed at improving (i.e., "humanizing") the glycosylation pattern of these recombinant proteins. Changes in medium composition as well as the co-expression of genes encoding enzymes involved in human glycosylation have been successfully employed (see, for example, Weikert, 1999).

[0026] While recombinant proteins similar to their human counterparts can be made in mammalian expression systems, it is currently not possible to make proteins with a human-like glycosylation pattern in lower eukaryotes (fungi and yeast). Although the core oligosaccharide structure transferred to a protein in the endoplasmic reticulum is basically identical in mammals and lower eukaryotes, substantial differences have been found in the subsequent processing reactions which occur in in the Golgi apparatus of fungi and mammals. In fact, even amongst different lower eukaryotes there exist a great variety of glycosylation structures. This has prevented the use of lower eukaryotes as hosts for the production of recombinant human glycoproteins despite otherwise notable advantages over mammalian expression systems, such as: (1) generally higher product titers, (2) shorter fermentation times, (3) having an alternative for proteins that are poorly expressed in mammalian cells, (4) the ability to grow in a chemically defined protein free medium and thus not requiring complex animal derived media components, (5) and the absence of viral, especially retroviral infections of such hosts.

[0027] Various methylotrophic yeasts such as Pichia pastoris, Pichia methanolica, and Hansenula polymorpha, have played particularly important roles as eukaryotic expression systems because they are able to grow to high cell densities and secrete large quantities of recombinant protein. However, as noted above, lower eukaryotes such as yeast do not glycosylate proteins like higher mammals. See for example, Martinet et al. (1998) Biotechnol Let. Vol. 20. No.12, which discloses the expression of a heterologous mannosidase in the endoplasmic reticulum (ER).

[0028] Chiba et al. (1998) have shown that S. cerevisiae can be engineered to provide structures ranging from Man.sub.8GlcNAc.sub.2 to Man.sub.5GlcNAc.sub.2 structures, by eliminating 1,6 mannosyltransferase (OCH1), 1,3 mannosyltransferase (MNN1) and a regulator of mannosylphosphatetransferase (MNN4) and by targeting the catalytic domain of .alpha.-1,2-mannosidase I from Aspergillus saitoi into the ER of S. cerevisiae using an ER retrieval sequence (Chiba, 1998). However, this attempt resulted in little or no production of the desired Man.sub.5GlcNAc.sub.2, e.g., one that was made in vivo and which could function as a substrate for GnT1 (the next step in making human-like glycan structures). Chiba et al. (1998) showed that P. pastoris is not inherently able to produce useful quantities (greater than 5%) of GlcNAcTransferase I accepting carbohydrate.

[0029] Maras and co-workers assert that in T. reesei "sufficient concentrations of acceptor substrate (i.e. Man.sub.5GlcNAc.sub.2) are present", however when trying to convert this acceptor substrate to GlcNAcMan.sub.5GlcNAc.sub.2 in vitro less than 2% were converted thereby demonstrating the presence of Man.sub.5GlcNAc.sub.2 structures that are not suitable precursors for complex N-glycan formation (Maras, 1997; Maras, 1999). To date no enabling disclosure exists, that allows for the production of commercially relevant quantities of GlcNAcMan.sub.5GlcNAc.sub.2 in lower eukaryotes.

[0030] It is therefore an object of the present invention to provide a system and methods for humanizing glycosylation of recombinant glycoproteins expressed in non-human host cells.

SUMMARY OF THE INVENTION

[0031] The present invention relates to host cells such as fungal strains having modified lipid-linked oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. A protein production method has been developed using (1) a lower eukaryotic host such as a unicellular or filamentous fungus, or (2) any non-human eukaryotic organism that has a different glycosylation pattern from humans, to modify the glycosylation composition and structures of the proteins made in a host organism ("host cell") so that they resemble more closely carbohydrate structures found in human proteins. The process allows one to obtain an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation by methods that are well established in the scientific literature and generally known to the artisan in the field of protein expression. As described herein, host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells have a GlcNAcMan.sub.3GlcNAc.sub.2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyl-transferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 is a schematic of the structure of the dolichyl pyrophosphate-linked oligosaccharide.

[0033] FIGS. 2A-2B show a schematic of the generation of GlcNAc.sub.2Man.sub.3GlcNAc.sub.2N-glycans from fungal host cells which are deficient in alg3, alg9 or alg 12 activities.

[0034] FIG. 3 is a schematic of processing reactions required to produce mammalian-type oligosaccharide structures in a fungal host cell with an alg3, och1 genotype.

[0035] FIGS. 4A-4F show S. cerevisiae Alg3 Sequence Comparisons (Blast).

[0036] SEQ ID NO:24 S. Cerevisiae (Query)

[0037] SEQ ID NO:25 S. Cerevisiae (Subject)

[0038] SEQ ID NO:26 S. Cerevisiae (Query)

[0039] SEQ ID NO:27 H. sapiens (Subject)

[0040] SEQ ID NO:28 S. Cerevisiae (Query)

[0041] SEQ ID NO:29Drosophilia virilis (Subject)

[0042] SEQ ID NO:30 S. Cerevisiae (Query)

[0043] SEQ ID NO:31 Drosophilia melanogaster (Subject)

[0044] FIG. 5 shows S. cerevisiae Alg 3 and Alg 3p Sequences

[0045] SEQ ID NO:32 DNA sequence

[0046] SEQ ID NO:33 amino acid sequence

[0047] FIG. 6 shows P. pastoris Alg 3 and Alg 3p Sequences

[0048] SEQ ID NO:34 DNA Sequence

[0049] SEQ ID NO:35 amino acid sequence

[0050] FIGS. 7A-7D show P. pastoris Alg 3 Sequence Comparisons (Blast)

[0051] SEQ ID NO:36 Pichia Pastoris (Query)

[0052] SEQ ID NO:37 S. Cerevisiae (Subject)

[0053] SEQ ID NO:38 (Query)

[0054] SEQ ID NO:39 Neurospora Crassa (Subject)

[0055] SEQ ID NO:40 Pichia Pastoris (Query)

[0056] SEQ ID NO:41 Schizosaccharomyces pombe (Subject)

[0057] SEQ ID NO:42 Pichia Pastoris

[0058] SEQ ID NO:43 Arabidopis thaliana

[0059] FIG. 8 shows K. lactis Alg 3 and Alg 3p Sequences

[0060] SEQ ID NO:44 DNA sequence

[0061] SEQ ID NO:45 amino acid sequence

[0062] FIG. 9 shows K. lactis Alg 3 Sequence Comparisons (Blast)

[0063] SEQ ID NO:46 K. lactis

[0064] SEQ ID NO:47 S. Cerevisiae

[0065] SEQ ID NO:48 K. lactis

[0066] SEQ ID NO:49 Arabidopis thaliana

[0067] FIG. 10 shows S. cerevisiae Alg 9 and Alg 9p Sequences

[0068] SEQ ID NO:50 S. Cerevisiae Alg 9 DNA

[0069] SEQ ID NO:51 S. Cerevisiae amino acid

[0070] FIG. 11 shows P. pastoris Alg 9 and Alg 9p Sequences

[0071] SEQ ID NO:52 Pichia Pastoris Alg 9 DNA

[0072] SEQ ID NO:53 Pichia Pastoris amino acid

[0073] FIGS. 12A-12C show P. pastoris Alg 9 Sequence Comparisons (Blast)

[0074] SEQ ID NO:54 Pichia Pastoris (Query)

[0075] SEQ ID NO:55 S. Cerevisiae (Subject)

[0076] SEQ ID NO:56 Pichia Pastoris (Query)

[0077] SEQ ID NO:57 Anopheles gambiae (Subject)

[0078] SEQ ID NO:58 Pichia Pastoris (Query)

[0079] SEQ ID NO: 59 S. pombe (Subject)

[0080] SEQ ID NO:60 Pichia Pastoris (Query)

[0081] SEQ ID NO:61 M. Musculus (Subject)

[0082] SEQ ID NO:62 Pichia Pastoris (Query)

[0083] SEQ ID NO:63 H. Sapiens (Subject)

[0084] FIG. 13 shows S. cerevisiae Alg 12 and Alg 12p Sequences

[0085] SEQ ID NO:64 S. Cerevisiae Alg 12 DNA

[0086] SEQ ID NO:65 S. Cerevisiae Alg 12 amino acid

[0087] FIG. 14 shows P. pastoris Alg 12 and Alg 12p Sequences

[0088] SEQ ID NO:66 Pichia Pastoris Alg 12 DNA

[0089] SEQ ID NO:67 S. Cerevisiae Alg 12 amino acid

[0090] FIGS. 15A-15B show P. pastoris Alg 12 Sequence Comparisons (Blast)

[0091] SEQ ID NO:68 Pichia Pastoris (Query)

[0092] SEQ ID NO:69 S. Cerevisiae (Subject)

[0093] SEQ ID NO:70 Pichia Pastoris (Query)

[0094] SEQ ID NO:71 S. pombe (Subject)

[0095] SEQ ID NO:72 Pichia Pastoris (Query)

[0096] SEQ ID NO:73 S. pombe (Subject)

[0097] FIG. 16 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris showing that the predominant N-glycan is GlcNAcMan.sub.5GlcNAc.sub.2.

[0098] FIG. 17 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris (FIG. 16) treated with P--N-hexosaminidase (peak corresponding to Man.sub.5GlcNAc.sub.2) to confirm that the predominant N-glycan of FIG. 16 is GlcNAcMan.sub.5GlcNAc.sub.2.

[0099] FIG. 18 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris alg3 deletion mutant showing that the predominant N-glycans are GlcNAcMan.sub.3GlcNAc.sub.2 and GlcNAcMan.sub.4GlcNAc.sub.2.

[0100] FIG. 19 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris alg3 deletion mutant treated with a1,2 mannosidase, showing that the GlcNAcMan.sub.4GlcNAc.sub.2 of FIG. 18 is converted to GlcNAcMan.sub.3GlcNAc.sub.2.

[0101] FIG. 20 is a MALDI-TOF-MS analysis of N-glycans of FIG. 19 treated with P--N-hexosaminidase (peak corresponding to Man.sub.3GlcNAc.sub.2) to confirm that the N-glycan of FIG. 19 is GlcNAcMan.sub.3GlcNAc.sub.2.

[0102] FIG. 21 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris alg3 deletion mutant treated with .alpha.1,2 mannosidase and GnTII, showing that the GlcNAcMan.sub.3GlcNAc.sub.2 of FIG. 19 is converted to GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.

[0103] FIG. 22 is a MALDI-TOF-MS analysis of N-glycans of FIG. 21 treated with P--N-hexosaminidase (peak corresponding to Man.sub.3GlcNAc.sub.2) to confirm that the N-glycan of FIG. 21 is GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.

[0104] FIG. 23 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris alg3 deletion mutant treated with .alpha.1,2 mannosidase and GnTII in the presence of UDP-galactose and .beta.1,4-galactosyltransferase, showing that the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 of FIG. 21 is converted to Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.

[0105] FIG. 24 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle 3 glycoprotein produced in a P. pastoris alg3 deletion mutant treated with .alpha.1,2 mannosidase and GnTII in the presence of UDP-galactose and .beta.1,4-galactosyltransferase, and further treated with CMP-N-acetylneuraminic acid and sialyltransferase, showing that the Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 is converted to NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2.

[0106] FIGS. 25A-25B show S. cerevisiae Alg6 and Alg 6p Sequences

[0107] SEQ ID NO:74 S. Cerevisiae DNA Alg 6

[0108] SEQ ID NO:75 S. Cerevisiae amino acid

[0109] SEQ ID NO:76 Pichia Pastoris DNA Alg 6

[0110] SEQ ID NO:77 Pichia Pastoris amino acid Alg 6

[0111] FIGS. 26A-26B show P. pastoris Alg6 and Alg 6p Sequences

[0112] SEQ ID NO:78 Pichia Pastoris (Query)

[0113] SEQ ID NO:79 S. Cerevisiae (Subject)

[0114] SEQ ID NO:80 Pichia Pastoris (Query)

[0115] SEQ ID NO:81 S. pombe (Subject)

[0116] SEQ ID NO:82 Pichia Pastoris (Query)

[0117] SEQ ID NO:83 D. melanogaster (Subject)

[0118] SEQ ID NO:84 Pichia Pastoris (Query)

[0119] SEQ ID NO:85 A. thaliana (Subject)

[0120] FIGS. 27A-27E show P. pastoris Alg 6 Sequence Comparisons (Blast)

[0121] FIG. 28 shows K. lactis Alg6 and Alg 6p Sequences

[0122] SEQ ID NO:86 K. lactis Alg 6 DNA

[0123] SEQ ID NO:87 K. lactis Alg 6 amino acid

[0124] FIGS. 29A-C show K. lactis Alg 6 Sequence Comparisons (Blast)

[0125] SEQ ID NO:88 K. lactis Alg 6 DNA

[0126] SEQ ID NO:89 S. Cerevisiae (Subject)

[0127] SEQ ID NO:90 K. lactis (Query)

[0128] SEQ ID NO:91 S. pombe (Subject)

[0129] SEQ ID NO:92 K. lactis (Query)

[0130] SEQ ID NO:93 A. thaliana (Subject)

[0131] SEQ ID NO:94 K. lactis (Query)

[0132] SEQ ID NO:95 H. Sapiens (Subject)

[0133] FIG. 30 Model of an IgG immunoglobulin. Heavy chain and light chain can be, based on similar secondary and tertiary structure, subdivided into domains. The two heavy chains (domains V.sub.H, C.sub.H1, C.sub.H2 and C.sub.H3) are linked through three disulfide bridges. The light chains (domains V.sub.L and C.sub.L) are linked by another disulfide bridge to the C.sub.H1 portion of the heavy chain and, together with the C.sub.H1 and V.sub.H fragments, make up the Fab region. Antigens bind to the terminal portion of the Fab region. Effector-functions, such as Fc-gamma-Receptor binding have been localized to the C.sub.H2 domain, just downstream of the hinge region and are influenced by N-glycosylation of asparagine 297 in the heavy chain.

[0134] FIG. 31 Schematic overview of a modular IgG1 expression vector.

[0135] FIG. 32 shows M. musculis GnT III Nucleic Acid And Amino Acid Sequences

[0136] SEQ ID NO:96 M. musculus DNA GnTIII

[0137] SEQ ID NO:97 M. musculus amino acid GnTIII

[0138] FIGS. 33A-33B show H. sapiens GnT IV Nucleic Acid And Amino Acid Sequences

[0139] SEQ ID NO:98 H. Sapiens DNA GnTIV

[0140] SEQ ID NO:99 H. Sapiens aa Gn TIV

[0141] FIGS. 34A-34B show M. musculis GnT VNucleic Acid And Amino Acid Sequences

[0142] SEQ ID NO:100 M. musculus DNA GnTV

[0143] SEQ ID NO:101 M. musculus aa GnTV

DETAILED DESCRIPTION OF THE INVENTION

[0144] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology, Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.; Handbook of Biochemistry: Section A Proteins Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins Vol II 1976 CRC Press; Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999). The nomenclatures used in connection with, and the laboratory procedures and techniques of, biochemistry and molecular biology described herein are those well known and commonly used in the art.

[0145] All publications, patents and other references mentioned herein are incorporated by reference.

[0146] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0147] As used herein, the term "N-glycan" refers to an N-linked oligosaccharide, e.g., one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-glycans have a common pentasaccharide core of Man.sub.3GlcNAc.sub.2 ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., fucose and sialic acid) that are added to the Man.sub.3GlcNAc.sub.2 ("Man3") core structure. N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid). A "high mannose" type N-glycan has five or more mannose residues. A "complex" type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a "trimannose" core. The "trimannose core" is the pentasaccharide core having a Man3 structure. Complex N-glycans may also have galactose ("Gal") residues that are optionally modified with sialic acid or derivatives ("NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc and core fucose ("Fuc"). A "hybrid" N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core.

[0148] Abbreviations used herein are of common usage in the art, see, e.g., abbreviations of sugars, above. Other common abbreviations include "PNGase", which refers to peptide N-glycosidase F (EC 3.2.2.18); "GlcNAc Tr (I-III)", which refers to one of three N-acetylglucosaminyltransferase enzymes; "NANA" refers to N-acetylneuraminic acid.

[0149] As used herein, the term "secretion pathway" refers to the assembly line of various glycosylation enzymes to which a lipid-linked oligosaccharide precursor and an N-glycan substrate are sequentially exposed, following the molecular flow of a nascent polypeptide chain from the cytoplasm to the endoplasmic reticulum (ER) and the compartments of the Golgi apparatus. Enzymes are said to be localized along this pathway. An enzyme X that acts on a lipid-linked glycan or an N-glycan before enzyme Y is said to be or to act "upstream" to enzyme Y; similarly, enzyme Y is or acts "downstream" from enzyme X.

[0150] As used herein, the term "alg X activity" refers to the enzymatic activity encoded by the "alg X" gene, and to an enzyme having that enzymatic activity encoded by a homologous gene or gene product (see below) or by an unrelated gene or gene product.

[0151] As used herein, the term "antibody" refers to a full antibody (consisting of two heavy chains and two light chains) or a fragment thereof. Such fragments include, but are not limited to, those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation, and those produced recombinantly, so long as the fragment remains capable of specific binding to an antigen. Among these fragments are Fab, Fab', F(ab')2, and single chain Fv (scFv) fragments. Within the scope of the term "antibody" are also antibodies that have been modified in sequence, but remain capable of specific binding to an antigen. Example of modified antibodies are interspecies chimeric and humanized antibodies; antibody fusions; and heteromeric antibody complexes, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Marasco (ed.), Intracellular Antibodies: Research and Disease Applications, Springer-Verlag New York, Inc. (1998) (ISBN: 3540641513), the disclosure of which is incorporated herein by reference in its entirety).

[0152] As used herein, the term "mutation" refers to any change in the nucleic acid or amino acid sequence of a gene product, e.g., of a glycosylation-related enzyme.

[0153] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. The term includes single and double stranded forms of DNA.

[0154] Unless otherwise indicated, a "nucleic acid comprising SEQ ID NO:X" refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ ID NO:X. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

[0155] An "isolated" or "substantially pure" nucleic acid or polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases, and genomic sequences with which it is naturally associated. The term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term "isolated" or "substantially pure" also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems.

[0156] However, "isolated" does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed "isolated" herein if a heterologous sequence (i.e., a sequence that is not naturally adjacent to this endogenous nucleic acid sequence) is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. By way of example, a non-native promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a human cell, such that this gene has an altered expression pattern. This gene would now become "isolated" because it is separated from at least some of the sequences that naturally flank it.

[0157] A nucleic acid is also considered "isolated" if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered "isolated" if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. An "isolated nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, a nucleic acid construct present as an episome. Moreover, an "isolated nucleic acid" can be substantially free of other cellular material, or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0158] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.

[0159] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, (herein incorporated by reference). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0160] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0161] Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. "Stringent hybridization conditions" and "stringent wash conditions" in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.

[0162] In general, "stringent hybridization" is performed at about 25.degree. C. below the thermal melting point (T.sub.m) for the specific DNA hybrid under a particular set of conditions. "Stringent washing" is performed at temperatures about 5.degree. C. lower than the T.sub.m, for the specific DNA hybrid under a particular set of conditions. The T.sub.m, is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., supra, page 9.51, hereby incorporated by reference. For purposes herein, "high stringency conditions" are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6.times.SSC (where 20.times.SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65.degree. C. for 8-12 hours, followed by two washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C. for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65.degree. C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.

[0163] The nucleic acids (also referred to as polynucleotides) of this invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[0164] The term "mutated" when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as "error-prone PCR" (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp. 11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2, pp. 28-33 (1992)); and "oligonucleotide-directed mutagenesis" (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R. T., et al., Science, 241, pp. 53-57 (1988)).

[0165] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0166] "Operatively linked" expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0167] The term "expression control sequence" as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0168] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

[0169] The term "peptide" as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

[0170] The term "polypeptide" encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

[0171] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) when it exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.

[0172] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0173] A "modified derivative" refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as .sup.125I, .sup.32P, .sup.35S, and .sup.3H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See Ausubel et al., 1992, hereby incorporated by reference.

[0174] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

[0175] The term "non-peptide analog" refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a "peptide mimetic" or a "peptidomimetic". See, e.g., Jones, (1992) Amino Acid and Peptide Synthesis, Oxford University Press; Jung, (1997) Combinatorial Peptide and Nonpeptide Libraries: A Handbook John Wiley; Bodanszky et al., (1993) Peptide Chemistry--A Practical Textbook, Springer Verlag; "Synthetic Peptides: A Users Guide", G. A. Grant, Ed, W. H. Freeman and Co., 1992; Evans et al. J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the invention may be used to produce an equivalent effect and are therefore envisioned to be part of the invention.

[0176] A "polypeptide mutant" or "mutein" refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. For instance, a mutein may have an increased or decreased neuron or NgR binding activity. In a preferred embodiment of the present invention, a MAG derivative that is a mutein (e.g., in MAG Ig-like domain 5) has decreased neuronal growth inhibitory activity compared to endogenous or soluble wild-type MAG.

[0177] A mutein has at least 70% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having 80%, 85% or 90% overall sequence homology to the wild-type protein. In an even more preferred embodiment, a mutein exhibits 95% sequence identity, even more preferably 97%, even more preferably 98% and even more preferably 99% overall sequence identity. Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.

[0178] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.

[0179] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2.sup.nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, .gamma.-carboxyglutamate, .epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0180] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences). In a preferred embodiment, a homologous protein is one that exhibits 60% sequence homology to the wild type protein, more preferred is 70% sequence homology. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence homology to the wild type protein. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence identity. As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0181] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, herein incorporated by reference).

[0182] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0183] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.

[0184] A preferred algorithm when comparing a inhibitory molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410; Gish and States (1993) Nature Genet. 3:266-272; Madden, T. L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. and Madden, T. L. (1997) Genome Res. 7:649-656), especially blastp or tblastn (Altschul et al., 1997). Preferred parameters for BLASTp are:

[0185] Expectation value: 10 (default)

[0186] Filter: seg (default)

[0187] Cost to open a gap: 11 (default)

[0188] Cost to extend a gap: 1 (default

[0189] Max. alignments: 100 (default)

[0190] Word size: 11 (default)

[0191] No. of descriptions: 100 (default)

[0192] Penalty Matrix: BLOWSUM62

[0193] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, herein incorporated by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.

[0194] "Specific binding" refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, "specific binding" discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction is at least about 10-7 M (e.g., at least about 10.sup.-8 M or 10.sup.-9 M).

[0195] The term "region" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0196] The term "domain" as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0197] As used herein, the term "molecule" means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0199] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Engineering or Selecting Hosts With Modified Lipid-Linked Oligosaccharides For The Generation of Human-like N-Glycans

[0200] The invention provides a method for producing a human-like glycoprotein in a non-human eukaryotic host cell. The method involves making or using a non-human eukaryotic host cell diminished or depleted in an alg gene activity (i.e., alg activities, including equivalent enzymatic activities in non-fungal host cells) and introducing into the host cell at least one glycosidase activity. In a preferred embodiment, the glycosidase activity is introduced by causing expression of one or more mannosidase activities within the host cell, for example, by activation of a mannosidase activity, or by expression from a nucleic acid molecule of a mannosidase activity, in the host cell.

[0201] In another embodiment, the method involves making or using a host cell diminished or depleted in the activity of one or more enzymes that transfer a sugar residue to the 1,6 arm of lipid-linked oligosaccharide precursors (FIG. 1). A host cell of the invention is selected for or is engineered by introducing a mutation in one or more of the genes encoding an enzyme that transfers a sugar residue (e.g., mannosylates) the 1,6 arm of a lipid-linked oligosaccharide precursor. The sugar residue is more preferably mannose, is preferably a glucose, GlcNAc, galactose, sialic acid, fucose or GlcNAc phosphate residue. In a preferred embodiment, the activity of one or more enzymes that mannosylate the 1,6 arm of lipid-linked oligosaccharide precursors is diminished or depleted. The method may further comprise the step of introducing into the host cell at least one glycosidase activity (see below).

[0202] In yet another embodiment, the invention provides a method for producing a human-like glycoprotein in a non-human host, wherein the glycoprotein comprises an N-glycan having at least two GlcNAcs attached to a trimannose core structure.

[0203] In each above embodiment, the method is directed to making a host cell in which the lipid-linked oligosaccharide precursors are enriched in Man.sub.XGlcNAc.sub.2 structures, where X is 3, 4 or 5 (FIG. 2). These structures are transferred in the ER of the host cell onto nascent polypeptide chains by an oligosaccharyl-transferase and may then be processed by treatment with glycosidases (e.g., .alpha.-mannosidases) and glycosyltransferases (e.g., GnT1) to produce N-glycans having GlcNAcMan.sub.XGlcNAc.sub.2 core structures, wherein X is 3, 4 or 5, and is preferably 3 (FIGS. 2 and 3). As shown in FIG. 2, N-glycans having a GlcNAcMan.sub.XGlcNAc.sub.2 core structure where X is greater than 3 may be converted to GlcNAcMan.sub.3GlcNAc.sub.2, e.g., by treatment with an .alpha.-1,3 and/or .alpha.-1,2-1,3 mannosidase activity, where applicable.

[0204] Additional processing of GlcNAcMan.sub.3GlcNAc.sub.2 by treatment with glycosyltransferases (e.g., GnTII) produces GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 core structures which may then be modified, as desired, e.g., by ex vivo treatment or by heterologous expression in the host cell of a set of glycosylation enzymes, including glycosyltransferases, sugar transporters and mannosidases (see below), to become human-like N-glycans. Preferred human-like glycoproteins which may be produced according to the invention include those which comprise N-glycans having seven or fewer, or three or fewer, mannose residues; comprise one or more sugars selected from the group consisting of galactose, GlcNAc, sialic acid, and fucose; and comprise at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man.

[0205] In one embodiment, the host cell has diminished or depleted Dol-P-Man:Man.sub.5GlcNAc.sub.2-PP-Dol Mannosyltransferase activity, which is an activity involved in the first mannosylation step from Man.sub.5GlcNAc.sub.2-PP-Dol to Man.sub.6GlcNAc.sub.2-PP-Dol at the luminal side of the ER (e.g., ALG3 FIG. 1; FIG. 2). In S. cerevisiae, this enzyme is encoded by the ALG3 gene. As described above, S. cerevisiae cells harboring a leaky alg3-1 mutation accumulate Man.sub.5GlcNAc.sub.2-PP-Dol and cells having a deletion in alg3 appear to transfer Man.sub.5GlcNAc.sub.2 structures onto nascent polypeptide chains within the ER. Accordingly, in this embodiment, host cells will accumulate N-glycans enriched in Man.sub.5GlcNAc.sub.2 structures which can then be converted to GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 by treatment with glycosidases (e.g., with .alpha.-1,2 mannosidase, .alpha.-1,3 mannosidase or .alpha.-1,2-1,3 mannosidase activities (FIG. 2).

[0206] As described in Example 1, degenerate primers were designed based on an alignment of Alg3 protein sequences from S. cerevisiae, D. melanogaster and humans (H. sapiens) (FIGS. 4 and 5), and were used to amplify a product from P. pastoris genomic DNA. The resulting PCR product was used as a probe to identify and isolate a P. pastoris genomic clone comprising an open reading frame (ORF) that encodes a protein having 35% overall sequence identity and 53% sequence similarity to the S. cerevisiae ALG3 gene (FIGS. 6 and 7). This P. pastoris gene is referred to herein as "PpALG3". The ALG3 gene was similarly identified and isolated from K. lactis (Example 1; FIGS. 8 and 9).

[0207] Thus, in another embodiment, the invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of at least forty-five, preferably at least 50, more preferably at least 60 and most preferably 75 or more nucleotide residues of the P. pastoris ALG 3gene (FIG. 6) and the K. lactis ALG 3gene (FIG. 8), and homologs, variants and derivatives thereof. The invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. Similarly, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided (P. pastoris and K. lactis ALG 3gene products are shown in FIGS. 6 and 8). In addition, also provided are vectors, including expression vectors, which comprise a nucleic acid molecule of the invention, as described further herein.

[0208] Using gene-specific primers, a construct was made to delete the PpALG3 gene from the genome of P. pastoris (Example 1). This strain was used to generate a host cell depleted in Dol-P-Man:Man.sub.5GlcNAc.sub.2-PP-Dol Mannosyltransferase activity and produce lipid-linked Man.sub.5GlcNAc.sub.2-PP-Dol precursors which are transferred onto nascent polypeptide chains to produce N-glycans having a Man.sub.5GlcNAc.sub.2 carbohydrate structure.

[0209] As described in Example 2, such a host cell may be engineered by expression of appropriate mannosidases to produce N-glycans having the desired Man.sub.3GlcNAc.sub.2 core carbohydrate structure. Expression of GnTs in the host cell (e.g., by targeting a nucleic acid molecule or a library of nucleic acid molecules as described below) enables the modified host cell to produce N-glycans having one or two GlcNAc structures attached to each arm of the Man3 core structure (i.e., GlcNAc.sub.1Man.sub.3GlcNAc.sub.2 or GlcNAc.sub.2Man.sub.3GlcNAc.sub.2; see FIG. 3). These structures may be processed further using the methods of the invention to produce human-like N-glycans on proteins which enter the secretion pathway of the host cell.

[0210] In another embodiment, the host cell has diminished or depleted dolichyl-P-Man:Man.sub.6GlcNAc2-PP-dolichyl .alpha.-1,2 mannosyltransferase activity, which is an .alpha.-1,2 mannosyltransferase activity involved in the mannosylation step converting Man.sub.6GlcNAc.sub.2-PP-Dol to Man.sub.7GlcNAc.sub.2-PP-Dol at the luminal side of the ER (see above and FIGS. 1 and 2). In S. cerevisiae, this enzyme is encoded by the ALG9 gene. Cells harboring an alg9 mutation accumulate Man.sub.6GlcNAc.sub.2-PP-Dol (FIG. 2) and transfer Man.sub.6GlcNAc.sub.2 structures onto nascent polypeptide chains within the ER. Accordingly, in this embodiment, host cells will accumulate N-glycans enriched in Man.sub.6GlcNAc.sub.2 structures which can then be processed down to core Man3 structures by treatment with .alpha.-1,2 and .alpha.-1,3 mannosidases (see FIG. 3 and Examples 3 and 4).

[0211] A host cell in which the alg9 gene (or gene encoding an equivalent activity) has been deleted is constructed (see, e.g., Example 3). Deletion of ALG9 (or ALG12; see below) creates a host cell which produces N-glycans with one or two additional mannoses, respectively, on the 1,6 arm (FIG. 2). In order to make the 1,6 core-mannose accessible to N-acetylglucosaminyltransferase II (GnTII) these mannoses have to be removed by glycosidase(s). ER mannosidase typically will remove the terminal 1,2 mannose on the 1,6 arm and subsequently Mannosidase II (alpha 1-3,6 mannosidase) or other mannosidases such as alpha 1,2, alpha 1,3 or alpha 1-2,3 mannosidases (e.g., from Xanthomonas manihotis; see Example 4) can act upon the 1,6 arm and subsequently GnTII can transfer an N-acetylglucosamine, resulting in GlcNAc.sub.2Man.sub.3 (FIG. 2).

[0212] The resulting host cell, which is depleted for alg9p activity, is engineered to express .alpha.-1,2 and .alpha.-1,3 mannosidase activity (from one or more enzymes, and preferably, by expression from a nucleic acid molecule introduced into the host cell and which expresses an enzyme targeted to a preferred subcellular compartment (see below). Example 4 describes the cloning and expression of one such enzyme from Xanthomonas manihotis.

[0213] In another embodiment, the host cell has diminished or depleted dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl .alpha.-1,6 mannosyltransferase activity, which is an .alpha.-1,6 mannosyltransferase activity involved in the mannosylation step converting Man.sub.7GlcNAc.sub.2-PP-Dol to Man.sub.8GlcNAc.sub.2-PP-Dol (which mannosylates the .alpha.-1,6 mannose on the 1,6 arm of the core mannose structure) at the luminal side of the ER (see above and FIGS. 1 and 2). In S. cerevisiae, this enzyme is encoded by the ALG12 gene. Cells harboring an alg12 mutation accumulate Man.sub.7GlcNAc.sub.2-PP-Dol (FIG. 2) and transfer Man.sub.7GlcNAc.sub.2 structures onto nascent polypeptide chains within the ER. Accordingly, in this embodiment, host cells will accumulate N-glycans enriched in Man.sub.7GlcNAc.sub.2 structures which can then be processed down to core Man3 structures by treatment with .alpha.-1,2 and .alpha.-1,3 mannosidases (see FIG. 3 and Examples 3 and 4).

[0214] As described above for alg9 mutant hosts, the resulting host cell, which is depleted for alg12p activity, is engineered to express .alpha.-1,2 and .alpha.-1,3 mannosidase activity (e.g., from one or more enzymes, and preferably, by expression from one or more nucleic acid molecules introduced into the host cell and which express an enzyme activity which is targeted to a preferred subcellular compartment (see below).

Engineering or Selecting Hosts Optionally Having Decreased Initiating .alpha.-1,6 Mannosyltransferase Activity

[0215] In a preferred embodiment, the method of the invention involves making or using a host cell which is both (a) diminished or depleted in the activity of an alg gene or in one or more activities that mannosylate N-glycans on the .alpha.-1,6 arm of the Man.sub.3GlcNAc.sub.2 ("Man3") core carbohydrate structure; and (b) diminished or depleted in the activity of an initiating .alpha.-1,6-mannosyltransferase, i.e., an initiation specific enzyme that initiates outer chain mannosylation (on the .alpha.-1,3 arm of the Man3 cores structure). In S. cerevisiae, this enzyme is encoded by the OCH1 gene. Disruption of the och1 gene in S. cerevisiae results in a phenotype in which N-linked sugars completely lack the poly-mannose outer chain. Previous approaches for obtaining mammalian-type glycosylation in fungal strains have required inactivation of OCH1 (see, e.g., Chiba, 1998). Disruption of the initiating .alpha.-1,6-mannosyltransferase activity in a host cell of the invention is optional, however (depending on the selected host cell), as the Och1p enzyme requires an intact Man.sub.8GlcNAc for efficient mannose outer chain initiation. Thus, the host cells selected or produced according to this invention, which accumulate lipid-linked oligosaccharides having seven or fewer mannose residues will, after transfer, produce hypoglycosylated N-glycans that will likely be poor substrates for Och1p (see, e.g., Nakayama, 1997).

Engineering or Selecting Hosts Having Increased Glucosyltransferase Activity

[0216] As discussed above, glucosylated oligosaccharides are thought to be transferred to nascent polypeptide chains at a much higher rate than their nonglucosylated counterparts. It appears that substrate recognition by the oligosaccharyltransferase complex is enhanced by addition of glucose to the antennae of lipid-linked oligosaccharides. It is thus desirable to create or select host cells capable of optimal glucosylation of the lipid-linked oligosaccharides. In such host cells, underglycosylation will be substantially decreased or even abolished, due to a faster and more efficient transfer of glucosylated Man.sub.5 structures onto the nascent polypeptide chain.

[0217] Accordingly, in another embodiment of the invention, the method is directed to making a host cell in which the lipid-linked N-glycan precursors are transferred efficiently to the nascent polypeptide chain in the ER. In a preferred embodiment, transfer is augmented by increasing the level of glucosylation on the branches of lipid-linked oligosaccharides which, in turn, will make them better substrates for oligosaccharyltransferase.

[0218] In one preferred embodiment, the invention provides a method for making a human-like glycoprotein which uses a host cell in which one or more enzymes responsible for glucosylation of lipid-linked oligosaccharides in the ER has increased activity. One way to enhance the degree of glucosylation of the lipid-linked oligosaccharides is to overexpress one or more enzymes responsible for the transfer of glucose residues onto the antennae of the lipid-linked oligosaccharide. In particular, increasing .alpha.-1,3 glucosyltransferase activity will increase the amount of glucosylated lipid-linked Man.sub.5 structures and will reduce or eliminate the underglycosylation of secreted proteins. In S. cerevisiae, this enzyme is encoded by the ALG6 gene.

[0219] Saccharomyces cerevisiae ALG6 and its human counterpart have been cloned (Imbach, 1999; Reiss, 1996). Due to the evolutionary conservation of the early steps of glycosylation, ALG6 loci are expected to be homologous between species and may be cloned based on sequence similarities by anyone skilled in the art. (The same holds true for cloning and identification of ALG8 and ALG10 loci from different species.) In addition, different glucosyltransferases from different species can then be tested to identify the ones with optimal activities.

[0220] The introduction of additional copies of an ALG6 gene and/or the expression of ALG6 under the control of a strong promoter, such as the GAPDH promoter, is one of several ways to increase the degree of glucosylated lipid-linked oligosaccharides. The ALG6 gene from P. pastoris is cloned and expressed (Example 5). ALG6 nucleic acid and amino acid sequences are show in FIG. 25 (S. cerevisiae) and FIG. 26 (P. pastoris). These sequences are compared to other eukaryotic ALG6 sequences in FIG. 27.

[0221] Accordingly, another embodiment of the invention provides a method to enhance the degree of glucosylation of lipid-linked oligosaccharides comprising the step of increasing alpha-1,3 glucosyltransferase activity in a host cell. The increase in activity may be achieved by overexpression of nucleic acid sequences encoding the activity, e.g., by operatively linking the nucleic acid encoding the activity with one or more heterologous expression control sequences. Preferred expression control sequences include transcription initiation, termination, promoter and enhancer sequences; RNA splice donor and polyadenylation signals; mRNA stabilizing sequences; ribosome binding sites; protein stabilizing sequences; and protein secretion sequences.

[0222] In another embodiment, the increase in alpha-1,3 glucosyltransferase activity is achieved by introducing a nucleic acid molecule encoding the activity on a multi-copy plasmid, using techniques well known to the skilled worker. In yet another embodiment, the degree of glucosylation of lipid-linked oligosaccharides comprising decreasing the substrate specificity of oligosaccharyl transferase activity in a host cell. This is achieved by, for example, subjecting at least one nucleic acid encoding the activity to a technique such as gene shuffling, in vitro mutagenesis, and error-prone polymerase chain reaction, all of which are well-known to one of skill in the art. Naturally, ALG8 and ALG10 can be overexpressed in a host cell and tested in a similar fashion.

[0223] Accordingly, in a preferred embodiment, the invention provides a method for making a human-like glycoprotein using a host cell which is engineered or selected so that one or more enzymes responsible for glucosylation of lipid-linked oligosaccharides in the ER has increased activity. In a more preferred embodiment, the invention uses a host cell having both (a) diminished or depleted in the activity of one or more alg gene activities or activities that mannosylate N-glycans on the .alpha.-1,6 arm of the Man.sub.3GlcNAc.sub.2 ("Man3") core carbohydrate structure and (b) engineered or selected so that one or more enzymes responsible for glucosylation of lipid-linked oligosaccharides in the ER has increased activity. The lipid-linked Man.sub.5 structure found in an alg3 mutant background, however, is not a preferred substrate for Alg6p. Accordingly, the skilled worker may identify Alg6p, Alg8p and Alg10p with an increased substrate specificity (Gibbs, 2001) e.g., by subjecting nucleic acids encoding such enzymes to one or more rounds of gene shuffling, error prone PCR, or in vitro mutagenesis approaches and selecting for increased substrate specificity in a host cell of interest, using molecular biology and genetic selection techniques well known to those of skill in the art. It will be appreciated by the skilled worker that such techniques for improving enzyme substrate specificities in a selected host strain are not limited to this particular embodiment of the invention but rather, may be used in any embodiment to optimize further the production of human-like N-glycans in a non-human host cell.

[0224] As described, once Man.sub.5 is transferred onto the nascent polypeptide chain, expression of suitable .alpha.-1,2-mannosidase(s), as provided by the present invention, will further trim Man.sub.5GlcNAc.sub.2 structures to yield the desired core Man.sub.3GlcNAc.sub.2 structures. .alpha.-1,2-mannosidases remove only terminal .alpha.-1,2-linked mannose residues and are expected to recognize the Man.sub.5GlcNAc.sub.2-Man.sub.7GlcNAc.sub.2 specific structures made in alg3, 9 and 12 mutant host cells and in host cells in which homologs to these genes are mutated.

[0225] As schematically presented in FIG. 3, co-expression of appropriate UDP-sugar-transporter(s) and -transferase(s) will cap the terminal .alpha.-1,6 and .alpha.-1,3 residues with GlcNAc, resulting in the necessary precursor for mammalian-type complex and hybrid N-glycosylation: GlcNAc.sub.2Man.sub.5GlcNAc.sub.2. The peptide-bound N-linked oligosaccharide chain GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (FIG. 3) then serves as a precursor for further modification to a mammalian-type oligosaccharide structure. Subsequent expression of galactosyl-tranferases and genetically engineering the capacity to transfer sialylic acid will produce a mammalian-type (e.g., human-like) N-glycan structure.

[0226] A desired host cell according to the invention can be engineered one enzyme or more than one enzyme at a time. In addition, a library of genes encoding potentially useful enzymes can be created, and a strain having one or more enzymes with optimal activities or producing the most "human-like" glycoproteins, selected by transforming target host cells with one or more members of the library. Lower eukaryotes that are able to produce glycoproteins having the core N-glycan Man.sub.3GlcNAc.sub.2 are particularly useful because of the ease of performing genetic manipulations, and safety and efficiency features. In a preferred embodiment, at least one further glycosylation reaction is performed, ex vivo or in vivo, to produce a human-like N-glycan. In a more preferred embodiment, active forms of glycosylating enzymes are expressed in the endoplasmic reticulum and/or Golgi apparatus of the host cell to produce the desired human-like glycoprotein.

Host Cells

[0227] A preferred non-human host cell of the invention is a lower eukaryotic cell, e.g., a unicellular or filamentous fungus, which is diminished or depleted in the activity of one or more alg gene activities (including an enzymatic activity which is a homolog or equivalent to an alg activity). Another preferred host cell of the invention is diminished or depleted in the activity of one or more enzymes (other than alg activities) that mannosylate the .alpha.-1,6 arm of a lipid-linked oligosaccharide structure.

[0228] While lower eukaryotic host cells are preferred, a wide variety of host cells having the aforementioned properties are envisioned as being useful in the methods of the invention. Plant cells, for instance, may be engineered to express a human-like glycoprotein according to the invention. Likewise, a variety of non-human, mammalian host cells may be altered to express more human-like glycoproteins using the methods of the invention. An appropriate host cell can be engineered, or one of the many such mutants already described in yeasts may be used. A preferred host cell of the invention, as exemplified herein, is a hypermannosylation-minus (OCH1) mutant in Pichia pastoris which has further been modified to delete the alg3 gene. Other preferred hosts are Pichia pastoris mutants having och1 and alg 9 or alg12 mutations.

Formation of Complex N-Glycans

[0229] The sequential addition of sugars to the modified, nascent N-glycan structure involves the successful targeting of glucosyltransferases into the Golgi apparatus and their successful expression. This process requires the functional expression, e.g., of GnT I, in the early or medial Golgi apparatus as well as ensuring a sufficient supply of UDP-GlcNAc (e.g., by expression of a UDP-GlcNAc transporter).

[0230] To characterize the glycoproteins and to confirm the desired glycosylation, the glycoproteins were purified, the N-glycans were PNGase-F released and then analyzed by MALDI-TOF-MS (Example 2). Kringle 3 domain of human plasminogen was used as the reporter protein. This soluble glycoprotein was produced in P. pastoris in an alg3, och1 knockout background (Example 2).

[0231] GlcNAcMan.sub.5GlcNAc.sub.2 was produced as the predominant N-glycan after addition of human GnT I, and K. lactis UDP-GlcNAc transporter in FIG. 16 (Example 2). The mass of this N-glycan is consistent with the mass of GlcNAcMan.sub.5GlcNAc.sub.2 at 1463 (m/z). To confirm the addition of the GlcNAc onto Man.sub.5GlcNAc.sub.2, a .beta.-N-hexosaminidase digest was performed, which revealed a peak at 1260 (m/z), consistent with the mass of Man.sub.5GlcNAc.sub.2 (FIG. 17).

[0232] The N-glycans from the alg3 och1 deletion in one strain PBP3 (Example 2) provided two distinct peaks at 1138 (m/z) and 1300 (m/z), which is consistent with structures GlcNAcMan.sub.3GlcNAc.sub.2 and GlcNAcMan.sub.4GlcNAc.sub.2 (FIG. 18). After an in vitro .alpha.1,2-mannosidase digestion for redundant mannoses, a peak eluted at 1138 (m/z), which is consistent with GlcNAcMan.sub.3GlcNAc.sub.2 (FIG. 19). To confirm the addition of the GlcNAc onto the Man.sub.3GlcNAc.sub.2 structure, a .beta.-N-hexosaminidase digest was performed, which revealed a peak at 934 (m/z), consistent with the mass of Man.sub.3GlcNAc.sub.2 (FIG. 20).

[0233] The addition of the second GlcNAc onto GlcNAcMan.sub.3GlcNAc.sub.2 is shown in FIG. 21. The peak at 1357 (m/z) corresponds to GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. To confirm the addition of the two GlcNAcs onto the core mannose structure Man.sub.3GlcNAc.sub.2, another .beta.-N-hexosaminidase digest was performed, which revealed a peak at 934 (m/z), consistent with the mass of Man.sub.3GlcNAc.sub.2 (FIG. 22). This is conclusive data displaying a complex-type glycoprotein made in yeast cells.

[0234] The in vitro addition of UDP-galactose and .beta. 1,4-galactosyltransferase onto the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 resulted in a peak at 1664 (m/z), which is consistent with the mass of Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (FIG. 23) Finally, the in vitro addition of CMP-N-acetylneuraminic acid and sialyltransferase resulted in a peak at 2248 (m/z), which is consistent with the mass of NANA.sub.2Gal.sub.2GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 (FIG. 24). The above data supports the use of non-mammalian host cells, which are capable of producing complex human-like glycoproteins.

Targeting of Glycosyl- and Galactosyl-Transferases to Specific Organelles.

[0235] Much work has been dedicated to revealing the exact mechanism by which these enzymes are retained and anchored to their respective organelle. Although complex, evidence suggests that, stem region, membrane spanning region and cytoplasmic tail individually or in concert direct enzymes to the membrane of individual organelles and thereby localize the associated catalytic domain to that locus.

[0236] The method by which active glycosyltransferases can be expressed and directed to the appropriate organelle such that a sequential order of reactions may occur, that leads to complex N-glycan formation, is as follows: [0237] (A) Establish a DNA library of regions that are known to encode proteins/peptides that mediate localization to a particular location in the secretory pathway (ER, Golgi and trans Golgi network). A limited selection of such enzymes and their respective location is shown in Table 1. These sequences may be selected from the host to be engineered as well as other related or unrelated organism. Generally such sequences fall into three categories: (1) N-terminal sequences encoding a cytosolic tail (ct), a transmembrane domain (tmd) and part of a somewhat more ambiguously defined stem region (sr), which together or individually anchor proteins to the inner (lumenal) membrane of the Golgi, (2) retrieval signals which are generally found at the C-terminus such as the HDEL or KDEL tetrapeptide, and (3) membrane spanning nucleotide sugar transporters, which are known to locate in the Golgi. In the first case, where the localization region consists of various elements (ct, tmd and sr) the library is designed such that the ct, the tmd and various parts of the stem region are represented. This may be accomplished by using PCR primers that bind to the 5' end of the DNA encoding the cytosolic region and employing a series of opposing primers that bind to various parts of the stem region. In addition one would create fusion protein constructs that encode sugar nucleotide transporters and known retrieval signals. [0238] (B) A second step involves the creation of a series of fusion protein constructs, that encode the above mentioned localization sequences and the catalytic domain of a particular glycosyltransferase cloned in frame to such localization sequence (e.g. GnT I, GalT, Fucosyltransferase or ST). In the case of a sugar nucleotide transporter fused to a catalytic domain one may design such constructs such that the catalytic domain (e.g. GnT I) is either at the N- or the C-terminus of the resulting polypeptide. The catalytic domain, like the localization sequence, may be derived from various different sources. The choice of such a catalytic domains may be guided by the knowledge of the particular environment in which the catalytic domain is to be active. For example, if a particular glycosyltransferase is to be active in the late Golgi, and all known enzymes of the host organism in the late Golgi have a pH optimum of 7.0, or the late Golgi is known to have a particular pH, one would try to select a catalytic domain that has maximum activity at that pH. Existing in vivo data on the activity of such enzymes, in particular hosts, may also be of use. For example, Schwientek and coworkers showed that GalT activity can be engineered into the Golgi of S. cerevisiae and showed that such activity was present by demonstrating the transfer of some Gal to existing GlcNAc.sub.2 in an alg mutant of S. cerevisiae. In addition, one may perform several rounds of gene shuffling or error prone PCR to obtain a larger diversity within the pool of fusion constructs, since it has been shown that single amino mutations may drastically alter the activity of glycoprotein processing enzymes (Romero et al., 2000). Full length sequences of glycosyltransferases and their endogenous anchoring sequence may also be used. In a preferred embodiment, such localization/catalytic domain libraries are designed to incorporate existing information on the sequential nature of glycosylation reactions in higher eukaryotes. In other words, reactions known to occur early in the course of glycoprotein processing require the targeting of enzymes that catalyze such reactions to an early part of the Golgi or the ER. For example, the trimming of Man.sub.8GlcNAc.sub.2 to Man.sub.5GlcNAc.sub.2 is an early step in complex N-glycan formation. Since protein processing is initiated in the ER and then proceeds through the early, medial and late Golgi, it is desirable to have this reaction occur in the ER or early Golgi. When designing a library for mannosidase I localization, one thus attempts to match ER and early Golgi targeting signals with the catalytic domain of mannosidase I.

[0239] Upon transformation of the host strain with the fusion construct library a selection process is used to identify which particular combination of localization sequence and catalytic domain in fact have the maximum effect on the carbohydrate structure found in such host strain. Such selection can be based on any number of assays or detection methods. They may be carried out manually or may be automated through the use of high troughput screening equipment.

[0240] In another example, GnT I activity is required for the maturation of complex N-glycans, because only after addition of GlcNAc to the terminal .alpha.1,3 mannose residue may further trimming of such a structure to the subsequent intermediate GlcNAcMan.sub.3GlcNAc.sub.2 structure occur. Mannosidase II is most likely not capable of removing the terminal .alpha.1,3- and .alpha.1,6- mannose residues in the absence of a terminal .beta.1,2-GlcNAc and thus the formation of complex N-glycans will not proceed in the absence of GnT I activity (Schachter, 1991). Alternatively, one may first engineer or select a strain that makes sufficient quantities of Man.sub.5GlcNAc.sub.2 as described in this invention by engineering or selecting a strain deficient in Alg3P activity. In the presence of sufficient UDP-GlcNAc transporter activity, as may be achieved by engineering or selecting a strain that has such UDP-GlcNAc transporter activity, GlcNAc can be added to the terminal .alpha.-1,3 residue by GnTI as in vitro a Man.sub.3 structure is recognized by by rat liver GnTI (Moller, 1992).

[0241] In another approach, one may incorporate the expression of a UDP-GlcNAc transporter into the library mentioned above such that the desired construct will contain: (1) a region by which the transformed construct is maintained in the cell (e.g. origin of replication or a region that mediates chromosomal integration), (2) a marker gene that allows for the selection of cells that have been transformed, including counterselectable and recyclable markers such as ura3 or T-urf13 (Soderholm, 2001) or other well characterized selection-markers (e.g., his4, bla, Sh ble etc.), (3) a gene encoding a UDP-GlcNAc transporter (e.g. from K. lactis, (Abeijon, 1996), or from H. sapiens (Ishida, 1996), and (4) a promotor activating the expression of the above mentioned localization/catalytic domain fusion construct library.

[0242] After transformation of the host with the library of fusion constructs described above, one may screen for those cells that have the highest concentration of terminal GlcNAc on the cell surface, or secrete the protein with the highest terminal GlcNAc content. Such a screen may be based on a visual method, like a staining procedure, the ability to bind specific terminal GlcNAc binding antibodies or lectins conjugated to a marker (such lectins are available from E.Y. Laboratories Inc., San Mateo, Calif.), the reduced ability of specific lectins to bind to terminal mannose residues, the ability to incorporate a radioactively labeled sugar in vitro, altered binding to dyes or charged surfaces, or may be accomplished by using a Fluorescence Assisted Cell Sorting (FACS) device in conjunction with a fluorophore labeled lectin or antibody (Guillen, 1998). It may be advantageous to enrich particular phenotypes within the transformed population with cytotoxic lectins. U.S. Pat. No. 5,595,900 teaches several methods by which cells with a desired extra-cellular carbohydrate structures may be identified. Repeatedly carrying out this strategy allows for the sequential engineering of more and more complex glycans in lower eukaryotes.

[0243] After transformation, one may select for transformants that allow for the most efficient transfer of GlcNAc by GlcNAc Transferase II from UDP-GlcNAc in an in vitro assay. This screen may be carried out by growing cells harboring the transformed library under selective pressure on an agar plate and transferring individual colonies into a 96-well microtiter plate. After growing the cells, the cells are centrifuged, the cells resuspended in buffer, and after addition of UDP-GlcNAc and GnT V, the release of UDP is determined either by HPLC or an enzyme linked assay for UDP. Alternatively, one may use radioactively labeled UDP-GlcNAc and GnT V, wash the cells and then look for the release of radioactive GlcNAc by N-actylglucosaminidase. All this may be carried manually or automated through the use of high throughput screening equipment.

[0244] Transformants that release more UDP, in the first assay, or more radioactively labeled GlcNAc in the second assay, are expected to have a higher degree of GlcNAcMan.sub.3GlcNAc.sub.2 (FIG. 3) on their surface and thus constitute the desired phenotype. Alternatively, one may any use any other suitable screen such as a lectin binding assay that is able to reveal altered glycosylation patterns on the surface of transformed cells. In this case the reduced binding of lectins specific to terminal mannoses may be a suitable selection tool. Galantus nivalis lectin binds specifically to terminal .alpha.-1,3 mannose, which is expected to be reduced if sufficient mannsosidase II activity is present in the Golgi. One may also enrich for desired transformants by carrying out a chromatographic separation step that allows for the removal of cells containing a high terminal mannose content. This separation step would be carried out with a lectin column that specifically binds cells with a high terminal mannose content (e.g. Galantus nivalis lectin bound to agarose, SIGMA.RTM., St. Louis, Mo.) over those that have a low terminal mannose content. In addition, one may directly create such fusion protein constructs, as additional information on the localization of active carbohydrate modifying enzymes in different lower eukaryotic hosts becomes available in the scientific literature. For example, the prior art teaches us that human .beta.1,4-GalTr can be fused to the membrane domain of MNT, a mannosyltransferase from S. cerevisiae, and localized to the Golgi apparatus while retaining its catalytic activity (Schwientek et al., 1995). If S. cerevisiae or a related organism is the host to be engineered one may directly incorporate such findings into the overall strategy to obtain complex N-glycans from such a host. Several such gene fragments in P. pastoris have been identified that are related to glycosyltransferases in S. cerevisiae and thus could be used for that purpose.

TABLE-US-00001 TABLE 1 Gene or Location of gene sequence Organism Function product MnsI S. cerevisiae mannosidase ER Och1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) Mnn2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) Mnn1 S. cerevisiae 1,3-mannosyltransferase Golgi (trans) Och1 P. pastoris 1,6-mannosyltransferase Golgi (cis) 2,6 ST H. sapiens 2,6-sialyltransferase trans-Golgi S. frugiperda network .beta.1,4 Gal T bovine milk UDP-Gal transporter Golgi Mnt1 S. cerevisiae 1,2-mannosyltransferase Golgi (cis) HDEL at S. cerevisiae retrieval signal ER C-terminus

Integration Sites

[0245] As one ultimate goal of this genetic engineering effort is a robust protein production strain that is able to perform well in an industrial fermentation process, the integration of multiple genes into the host (e.g., fungal) chromosome involves careful planning. The engineered strain will most likely have to be transformed with a range of different genes, and these genes will have to be transformed in a stable fashion to ensure that the desired activity is maintained throughout the fermentation process. Any combination of the following enzyme activities will have to be engineered into the fungal protein expression host: sialyltransferases, mannosidases, fucosyltransferases, galactosyltransferases, glucosyltransferases,

[0246] GlcNAc transferases, ER and Golgi specific transporters (e.g. syn and antiport transporters for UDP-galactose and other precursors), other enzymes involved in the processing of oligosaccharides, and enzymes involved in the synthesis of activated oligosaccharide precursors such as UDP-galactose, CMP-N-acetylneuraminic acid. At the same time, a number of genes which encode enzymes known to be characteristic of non-human glycosylation reactions, will have to be deleted. Such genes and their corresponding proteins have been extensively characterized in a number of lower eukaryotes (e.g. S. cerevisiae, T. reesei, A. nidulans etc.), thereby providing a list of known glycosyltransferases in lower eukaryotes, their activities and their respective genetic sequence. These genes are likely to be selected from the group of mannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 in S. cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KRE family from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S. cerevisiae), mannosylphosphate transferases (MNN4 and MNN6 from S. cerevisiae) and additional enzymes that are involved in aberrant i.e. non human glycosylation reactions. Many of these genes have in fact been deleted individually giving rise to viable phenotypes with altered glycosylation profiles. Examples are shown in Table 2:

TABLE-US-00002 TABLE 2 Structure wild Structure Strain Mutant type mutant Authors Schizosaccharomyces OCH1 Mannan (i.e. Man.sub.8GlcNAc.sub.2 Yoko-o et al., 2001 pombe Man.sub.>9GlcNAc.sub.2) S. cerevisiae OCH1, Mannan (i.e. Man.sub.8GlcNAc.sub.2 Nakanishi-Shindo MNN1 Man.sub.>9GlcNAc.sub.2) et al,. 1993 S. cerevisiae OCH1, Mannan (i.e. Man.sub.8GlcNAc.sub.2 Chiba et al., 1998 MNN1, Man.sub.>9GlcNAc.sub.2) MNN4

As any strategy to engineer the formation of complex N-glycans into a lower eukaryote involves both the elimination as well as the addition of glycosyltransferase activities, a comprehensive scheme will attempt to coordinate both requirements. Genes that encode enzymes that are undesirable serve as potential integration sites for genes that are desirable. For example, 1,6 mannosyltransferase activity is a hallmark of glycosylation in many known lower eukaryotes. The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been cloned from S. cerevisiae and mutations in the gene give raise to a viable phenotype with reduced mannosylation. The gene locus encoding alpha-1,6 mannosyltransferase activity therefor is a prime target for the integration of genes encoding glycosyltransferase activity. In a similar manner, one can choose a range of other chromosomal integration sites that, based on a gene disruption event in that locus, are expected to: (1) improve the cells ability to glycosylate in a more human like fashion, (2) improve the cells ability to secrete proteins, (3) reduce proteolysis of foreign proteins and (4) improve other characteristics of the process that facilitate purification or the fermentation process itself.

Providing Sugar Nucleotide Precursors

[0247] A hallmark of higher eukaryotic glycosylation is the presence of galactose, fucose, and a high degree of terminal sialic acid on glycoproteins. These sugars are not generally found on glycoproteins produced in yeast and filamentous fungi and the method discussed above allows for the engineering of strains that localize glycosyltransferase in the desired organelle. Formation of complex N-glycan synthesis is a sequential process by which specific sugar residues are removed and attached to the core oligosaccharide structure. In higher eukaryotes, this is achieved by having the substrate sequentially exposed to various processing enzymes. These enzymes carry out specific reactions depending on their particular location within the entire processing cascade. This "assembly line" consists of ER, early, medial and late Golgi, and the trans Golgi network all with their specific processing environment. To recreate the processing of human glycoproteins in the Golgi and ER of lower eukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases, phosphatases and transporters) have to be expressed and specifically targeted to these organelles, and preferably, in a location so that they function most efficiently in relation to their environment as well as to other enzymes in the pathway.

[0248] Several individual glycosyltransferases have been cloned and expressed in S. cerevisiae (GalT, GnT I), Aspergillus nidulans (GnT I) and other fungi, without however demonstrating the desired outcome of "humanization" on the glycosylation pattern of the organisms (Yoshida, 1995; Schwientek, 1995; Kalsner, 1995). It was speculated that the carbohydrate structure required to accept sugars by the action of such glycosyltransferases was not present in sufficient amounts. While this most likely contributed to the lack of complex N-glycan formation, there are currently no reports of a fungus supplying a Man.sub.5GlcNAc.sub.2 structure, having GnT I activity and having UDP-Gn transporter activity engineered into the fungus. It is the combination of these three biochemical events that are required for hybrid and complex N-glycan formation.

[0249] In humans, the full range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generally synthesized in the cytosol and transported into the Golgi, where they are attached to the core oligosaccharide by glycosyltransferases. To replicate this process in lower eukaryotes, sugar nucleoside specific transporters have to be expressed in the Golgi to ensure adequate levels of nucleoside sugar precursors (Sommers, 1981; Sommers, 1982; Perez, 1987). A side product of this reaction is either a nucleoside diphosphate or monophosphate. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphospho nucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction appears to be important for efficient glycosylation, as GDPase from S. cerevisiae has been found to be necessary for mannosylation. However, the enzyme only has 10% of the activity towards UDP (Berninsone, 1994). Lower eukaryotes often do not have UDP specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.

[0250] Schizosaccharomyces pombe, a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) was found to have specific UDPase activity further suggesting the requirement for such an enzyme (Berninsone et al., 1994). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product is important in order to prevent glycosyltransferase inhibition in the lumen of the Golgi (Khatara et al., 1974). Thus, one may need to provide for the removal of UDP, which is expected to accumulate in the Golgi of such an engineered strains (Berninsone, 1995; Beaudet, 1998).

[0251] In another example, 2,3 sialyltransferase and 2,6 sialyltransferase cap galactose residues with sialic acid in the trans-Golgi and TGN of humans leading to a mature form of the glycoprotein. To reengineer this processing step into a metabolically engineered yeast or fungus will require (1) 2,3-sialyltransferase activity and (2) a sufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi of yeast. To obtain sufficient 2,3-sialyltransferase activity in the late Golgi, the catalytic domain of a known sialyltransferase (e.g. from humans) has to be directed to the late Golgi in fungi (see above). Likewise, transporters have to be engineered to that allow the transport of CMP-N-acetyl neuraminic acid into the late Golgi. There is currently no indication that fungi synthesize sufficient amounts of CMP-N-acetyl neuraminic acid, not to mention the transport of such a sugar-nucleotide into the Golgi. Consequently, to ensure the adequate supply of substrate for the corresponding glycosyltransferases, one has to metabolically engineer the production of CMP-sialic acid into the fungus.

Methods for Providing Sugar Nucleotide Precursors to the Golgi Apparatus:

UDP-N-acetyl-glucosamine

[0252] The cDNA of human UDP-N-acetylglucosamine transporter, which was recognized through a homology search in the expressed sequence tags database (dbEST) was cloned by Ishida and coworkers (Ishida, 1999). Guillen and coworkers have cloned the mammalian Golgi membrane transporter for UDP-N-acetylglucosamine by phenotypic correction with cDNA from canine kidney cells (MDCK) of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar (Guillen, 1998). Their results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane (Guillen, 1998).

GDP-Fucose

[0253] The rat liver Golgi membrane GDP-fucose transporter has been identified and purified by Puglielli, L. and C. B. Hirschberg (Puglielli, 1999). The corresponding gene has not been identified however N-terminal sequencing can be used for the design of oligonucleotide probes specific for the corresponding gene. These oligonucleotides can be used as probes to clone the gene encoding for GDP-fucose transporter.

UDP-Galactose

[0254] Two heterologous genes, gmal2(+) encoding alpha 1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomyces pombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter, have been functionally expressed in S. cerevisiae to examine the intracellular conditions required for galactosylation. Correlation between protein galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter, rather than alpha 1,2 GalT played a key role for efficient galactosylation in S. cerevisiae (Kainuma, 1999). Likewise a UDP-galactose transporter from S. pombe was cloned (Aoki, 1999; Segawa, 1999).

CMP-N-acetylneuraminic Acid (CMP-Sialic Acid)

[0255] Human CMP-sialic acid transporter (hCST) has been cloned and expressed in Lec 8 CHO cells (Aoki, 1999; Eckhardt, 1997). The functional expression of the murine CMP-sialic acid transporter was achieved in Saccharomyces cerevisiae (Berninsone, 1997). Sialic acid has been found in some fungi, however it is not clear whether the chosen host system will be able to supply sufficient levels of CMP-Sialic acid. Sialic acid can be either supplied in the medium or alternatively fungal pathways involved in sialic acid synthesis can also be integrated into the host genome.

Diphosphatases

[0256] When sugars are transferred onto a glycoprotein, either a nucleoside diphosphate or monophosphate, is released from the sugar nucleotide precursors. While monophosphates can be directly exported in exchange for nucleoside triphosphate sugars by an antiport mechanism, diphospho nucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphates and inorganic phosphate prior to being exported. This reaction appears to be important for efficient glycosylation, as GDPase from S. cerevisiae has been found to be necessary for mannosylation. However, the enzyme only has 10% of the activity towards UDP (Berninsone, 1994). Lower eukayotes often do not have UDP specific diphosphatase activity in the Golgi since they do not utilize UDP-sugar precursors for glycoprotein synthesis in the Golgi.

[0257] Schizosaccharomyces pombe, a yeast found to add galactose residues to cell wall polysaccharides (from UDP-galactose) was found to have specific UDPase activity further suggesting the requirement for such an enzyme (Berninsone, 1994). UDP is known to be a potent inhibitor of glycosyltransferases and the removal of this glycosylation side product is important in order to prevent glycosyltransferase inhibition in the lumen of the Golgi (Khatara et al. 1974).

Expression of GnTs to Produce Complex N-Glycans

Expression of GnT-III to Boost Antibody Functionality

[0258] The addition of an N-acetylglucosamine to the GlcNAc.sub.1Man.sub.3GlcNAc.sub.2 structure by N-acetylglucosaminyltransferases II and III yields a so-called bisected N-glycan GlcNAc.sub.3Man.sub.3GlcNAc.sub.2 (FIG. 3). This structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC) (Umana et al. 1999). Re-engineering glycoforms of immunoglobulins expressed by mammalian cells is a tedious and cumbersome task. Especially in the case of GnTIII, where over-expression of this enzyme has been implicated in growth inhibition, methods involving regulated (inducible) gene expression had to be employed to produce immunoglobulins with bisected N-glycans (Umana et al 1999a, 1999b).

[0259] Accordingly, in another embodiment, the invention provides systems and methods for producing human-like N-glycans having bisecting N-acetylglucosamine (GlcNAcs) on the core mannose structure. In a preferred embodiment, the invention provides a system and method for producing immunoglobulins having bisected N-glycans. The systems and methods described herein will not suffer from previous problems, e.g., cytotoxicity associated with overexpression of GnTIII or ADCC, as the host cells of the invention are engineered and selected to be viable and preferably robust cells which produce N-glycans having substantially modified human-type glycoforms such as GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. Thus, addition of a bisecting N-acetylglucosamine in a host cell of the invention will have a negligible effect on the growth-phenotype or viability of those host cells.

[0260] In addition, previous work (Umana) has shown that there is no linear correlation between GnTIII expression levels and the degree of ADCC. Finding the optimal expression level in mammalian cells and maintaining it throughout an FDA approved fermentation process seems to be a challenge. However, in cells of the invention, such as fungal cells, finding a promoter of appropriate strength to establish a robust, reliable and optimal GnTIII expression level is a comparatively easy task for one of skill in the art.

[0261] A host cell such as a yeast strain capable of producing glycoproteins with bisecting N-glycans is engineered according to the invention, by introducing into the host cell a GnTIII activity (Example 6). Preferably, the host cell is transformed with a nucleic acid that encodes GnTIII (see, e.g., FIG. 32) or a domain thereof having enzymatic activity, optionally fused to a heterologous cell signal targeting peptide (e.g., using the libraries and associated methods of the invention.) Host cells engineereded to express GnTIII will produce higher antibody titers than mammalian cells are capable of They will also produce antibodies with higher potency with respect to ADCC.

[0262] Antibodies produced by mammalian cell lines transfected with GnTIII have been shown to be as effective as antibodies produced by non-transfected cell-lines, but at a 10-20 fold lower concentration (Davies et al. 2001). An increase of productivity of the production vehicle of the invention over mammalian systems by a factor of twenty, and a ten-fold increase of potency will result in a net-productivity improvement of two hundred. The invention thus provides a system and method for producing high titers of an antibody having high potency (e.g., up to several orders of magnitude more potent than what can currently be produced). The system and method is safe and provides high potency antibodies at low cost in short periods of time. Host cells engineered to express GnT III according to the invention produce immunoglobulins having bisected N-glycans at rates of at least 50 mg/liter/day to at least 500 mg/liter/day. In addition, each immunoglobulin (Ig) molecule (comprising bisecting GlcNAcs) is more potent than the same Ig molecule produced without bisecting GlcNAcs.

Cloning and Expression of GnT-IV and GnT-V

[0263] All branching structures in complex N-glycans are synthesized on a common core-pentasaccharide (Man.sub.3GlcNAc.sub.2 or Man alpha1-6(Man alpha1-3)Man beta1-4 GlcNAc beta1-4 GlcNAc beta1-4 or Man.sub.3GlcNAc.sub.2) by N-acetylglucosamine transferases (GnTs)-I to -VI (Schachter H et al. (1989) Methods Enzymo;179:351-97). Current understanding of the biosynthesis of more highly branched N-glycans suggests that after the action of GnTII (generation of GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 structures) GnTIV transfers GlcNAc from UDP-GlcNAc in beta1,4 linkage to the Man alpha1,3 Man beta1,4 arm of GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 N-glycans (Allen S D et al. (1984) J Biol Chem. June 10; 259(11):6984-90; and Glceson P A and Schachter H. J (1983); J. Biol Chem 25;258(10):6162-73) resulting in a triantennary agalacto sugar chain. This N-glycan (GlcNAc beta1-2 Man alpha1-6(GlcNAc beta1-2 Man alpha1-3) Man beta1-4 GlcNAc beta 1-4 GlcNAc beta1,4 Asn) is a common substrate for GnT-III and -V, leading to the synthesis of bisected, tri-and tetra-antennary structures. Where the action of GnTIII results in a bisected N-glycan and where GnTV catalyzes the addition of beta 1-6GlcNAc to the alpha 1-6 mannosyl core, creating the beta 1-6 branch. Addition of galactose and sialic acid to these branches leads to the generation of a fully sialylated complex N-glycan.

[0264] Branched complex N-glycans have been implicated in the physiological activity of therapeutic proteins, such as human erythropoietin (hEPO). Human EPO having bi-antennary structures has been shown to have a low activity, whereas hEPO having tetra-antennary structures resulted in slower clearance from the bloodstream and thus in higher activity (Misaizu T et al. (1995) Blood December 1; 86(11):4097-104).

[0265] With DNA sequence information, the skilled worker can clone DNA molecules encoding GnT IV and/or V activities (Example 6; FIGS. 33 and 34). Using standard techniques well-known to those of skill in the art, nucleic acid molecules encoding GnT IV or V (or encoding catalytically active fragments thereof) may be inserted into appropriate expression vectors under the transcriptional control of promoters and other expression control sequences capable of driving transcription in a selected host cell of the invention, e.g., a fungal host such as Pichia sp., Kluyveromyces sp. and Aspergillus sp., as described herein, such that one or more of these mammalian GnT enzymes may be actively expressed in a host cell of choice for production of a human-like complex glycoprotein.

[0266] The following are examples which illustrate the compositions and methods of this invention. These examples should not be construed as limiting: the examples are included for the purposes of illustration only.

EXAMPLE 1

Identification, Cloning and Deletion of the ALG3 Gene in P. pastoris and K. lactis.

[0267] Degenerate primers were generated based on an alignment of Alg3 protein sequences from S. cerevisiae, H. sapiens, and D. melanogaster and were used to amplify an 83 by product from P. pastoris genomic DNA: [0268] 5'-GGTGTTTTGTTTTCTAGATCTTTGCAYTAYCARTT-3' (SEQ ID NO. 1) and [0269] 5'-AGAATTTGGTGGGTAAGAATTCCARCACCAYTCRTG-3' (SEQ ID NO. 2). The resulting PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and seqence analysis revealed homology to known ALG3/RHK1/NOT56 homologs (Genbank NC.sub.--001134.2, AF309689, NC.sub.--003424.1). Subsequently, 1929 by upstream and 2738 by downstream of the initial PCR product were amplified from a P. pastoris genomic DNA library (Boehm, T. Yeast 1999 May; 15(7):563-72) using the internal oligonucleotides [0270] 5'-CCTAAGCTGGTATGCGTTCTCTTTGCCATATC-3' (SEQ ID NO. 3) and [0271] 5'-GCGGCATAAACAATAATAGATGCTATAAAG-3' (SEQ ID NO. 4) along with T3 [0272] (5'-AATTAACCCTCACTAAAGGG-3') (SEQ ID NO. 5) and T7 (5'-GTAA TACGACTCACTATAGGGC-3') (SEQ ID NO. 6). (Integrated DNA Technologies, Coralville, Iowa) in the backbone of the library bearing plasmid lambda ZAP II (Stratagene, La Jolla, Calif.). The resulting fragments were cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. From this sequence, a 1395 bp ORF was identified that encodes a protein with 35% identity and 53% similarity to the S. cerevisiae ALG3 gene (using BLAST programs). The gene was named PpALG3.

[0273] The sequence of PpALG3was used to create a set of primers to generate a deletion construct of the PpALG3 gene by PCR overlap (Davidson et al, 2002 Microbiol. 148(Pt 8):2607-15). Primers below were used to amplify 1 kb regions 5' and 3' of the PpALG3 ORF and the KAN.sup.R gene, respectively:

TABLE-US-00003 RCD142 (SEQ ID NO. 7) (5'-CCACATCATCCGTGCTACATATAG-3'), RCD144 (SEQ ID NO. 8) (5'-ACGAGGCAAGCTAAACAGATCTCGAAGTATCGAGGGTTAT CCAG-3'), RCD145 (SEQ ID NO. 9) (5'-CCATCCAGTGTCGAAAACGAGCCAATGGTTCATGTCTATA AATC-3'), RCD147 (SEQ ID NO. 10) (5'-AGCCTCAGCGCCAACAAGCGATGG-3'), RCD143 (SEQ ID NO. 11) (5'-CTGGATAACCCTCGATACTTCGAGATCTGTTTAGCTTGCC TCGT-3'), and RCD146 (SEQ ID NO. 12) (5'-GATTTATAGACATGAACCATTGGCTCGTTTTCGACACTGG ATGG-3').

Subsequently, primers RCD142 and RCD147 were used to overlap the three resulting PCR products into a single 3.6 kb alg3::KAN.sup.R deletion allele. Identification, Cloning and Deletion of the ALG3 Gene in K. lactis.

[0274] The ALG3p sequences from S. cerevisiae, Drosophila melanogaster, Homo sapiens etc. were aligned with K. lactis sequences (PENDANT EST database). Regions of high homology that were in common homologs but distinct in exact sequence from the homologs were used to create pairs of degenerate primers that were directed against genomic DNA from the K. lactis strain MG1/2 (Bianchi et al, 1987). In the case of ALG3, PCR amplification with primers KAL-1 (5'-ATCCTTTACCGATGCTGTAT-3') (SEQ ID NO. 13) and KAL-2 (5'-ATAACAGTATGTGTTACACGCGTGTAG-3') (SEQ ID NO. 14) resulted in a product that was cloned and sequenced and the predicted translation was shown to have a high degree of homology to Alg3p proteins (>50% to S. cerevisiae Alg3p).

[0275] The PCR product was used to probe a Southern blot of genomic DNA from K. lactis strain (MG1/2) with high stringency (Sambrook et al, 1989). Hybridization was observed in a pattern consistent with a single gene. This Southern blot was used to map the genomic loci. Genomic fragments were cloned by digesting genomic DNA and ligating those fragments in the appropriate size-range into pUC19 to create a K. lactis subgenomic library. This subgenomic library was transformed into E. coli and several hundred clones were tested by colony PCR, using primers KAL-1 and KAL-2. The clones containing the predicted KlALG3 and KlALG61 genes were sequenced and open reading frames identified.

[0276] Primers for construction of an alg3::NAT.sup.R deletion allele, using a PCR overlap method (Davidson et al, 2002), were designed and the resulting deletion allele was transformed into two K. lactis strains and NAT-resistant colonies selected. These colonies were screened by PCR and transformants were obtained in which the ALG3 ORF was replaced with the och1::NAT.sup.R mutant allele.

EXAMPLE 2

Generation of an alg3/och1 Mutant Strain Expressing an .alpha.-1,2-Mannosidase, GnT1 and GnTII for Production of a Human-Like Glycoprotein.

[0277] The 1215 bp open reading frame of the P. pastoris OCH1 gene as well as 2685 bp upstream and 1175 bp downstream was amplified by PCR (B. K. Choi et al., submitted to Proc. Natl. Acad. Sci. USA 2002; see also WO 02/00879; each of which is incorporated herein by reference), cloned into the pCR2.1-TOPO vector (Invitrogen) and designated pBK9. To create an och1 knockout strain containing multiple auxotrophic markers, 100 .mu.g of pJN329, a plasmid containing an och1::URA3 mutant allele flanked with SfiI restriction sites was digested with SfiI and used to transform P. pastoris strain JC308 (Cereghino et al. Gene 263 (2001) 159-169) by electroporation. Following incubation on defined medium lacking uracil for 10 days at room temperature, 1000 colonies were picked and re-streaked. URA.sup.+ clones that were unable to grow at 37.degree. C., but grew at room temperature, were subjected to colony PCR to test for the correct integration of the och1::URA3 mutant allele. One clone that exhibited the expected PCR pattern was designated YJN153. The Kringle 3 domain of human plasminogen (K3) was used as a model protein. A Neo.sup.R marked plasmid containing the K3 gene was transformed into strain YJN153 and a resulting strain, expressing K3, was named BK64-1 (B. K. Choi et al, submitted to Proc. Natl. Acad. Sci. USA 2002).

[0278] Plasmid pPB 103, containing the Kluyveromyces lactis MNN2-2 gene, encoding a Golgi UDP-N-acetylglucosamine transporter was constructed by cloning a blunt BglII-HindIII fragment from vector pDL02 (Abeijon et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) into BglII and BamHI digested and blunt ended pBLADE-SX containing the P. pastoris ADE1 gene (Cereghino et al. (2001) Gene 263:159-169). This plasmid was linearized with EcoNI and transformed into strain BK64-1 by electroporation and one strain confirmed to contain the MNN2-2 by PCR analysis was named PBP1.

[0279] A library of mannosidase constructs was generated, comprising in-frame fusions of the leader domains of several type I or type II membrane proteins from S. cerevisiae and P. pastoris fused with the catalytic domains of several .alpha.-1,2-mannosidase genes from human, mouse, fly, worm and yeast sources (see, e.g., WO02/00879, incorporated herein by reference). This library was created in a P. pastoris HIS4 integration vector and screened by linearizing with SalI, transforming by electroporation into strain PBP 1, and analyzing the glycans released from the K3 reporter protein. One active construct chosen was a chimera of the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fused with a N-terminal deletion of the mouse .alpha.-1,2-mannosidase IA (MmMannIA) gene, which was missing the 187 nucleotides. A P. pastoris strain expressing this construct was named PBP2.

[0280] A library of GnTI constructs was generated, comprising in-frame fusions of the same leader library with the catalytic domains of GnTI genes from human, worm, frog and fly sources (WO 02/00879). This library was created in a P. pastoris ARG4 integration vector and screened by linearizing with AatII, transforming by electroporation into strain PBP2, and analyzing the glycans released from K3. One active construct chosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 gene fused to a deletion of the human GnTI gene, which was missing the first 154 bp. A P. pastoris strain expressing this construct was named PBP3.

[0281] Subsequently, a P. pastoris alg3::KAN.sup.R deletion construct was generated as described above. Approximately 5 .mu.g of the resulting PCR product was transformed into strain PBP3 and colonies were selected on YPD medium containing 200 .mu.g/ml G418. One strain out of 20 screened by PCR was confirmed to contain the correct integration of the alg3::KAN.sup.R mutant allele and lack the wild-type allele. This strain was named RDP27.

[0282] Finally, a library of GnTII constructs was generated, which was comprised of in-frame fusions of the leader library with the catalytic domains of GnTII genes from human and rat sources (WO 02/00879). This library was created in a P. pastoris integration vector containing the NST.sup.R gene conferring resistance to the drug nourseothricin. The library plasmids were linearized with EcoRI, transformed into strain RDP27 by electroporation, and the resulting strains were screened by analysis of the released glycans from purified K3.

Materials

[0283] MOPS, sodium cacodylate, manganese chloride, UDP-galactose and CMP-N-acetylneuraminic acid were from SIGMA.RTM.. TFA was from ALDRICH.RTM.. Recombinant rat .alpha.2,6-sialyltransferase from Spodoptera frugiperda and .beta.1,4-galactosyltransferase from bovine milk were from CALBIOCHEM.RTM.. Protein N-glycosidase F, mannosidases, and oligosaccharides were from GLYKO.RTM. (San Rafael, Calif.). DEAE TOYOPEARL.RTM. resin was from TosoHaas. Metal chelating "HisBind" resin was from Novagen (Madison, Wis.). 96-well lysate-clearing plates were from Promega (Madison, Wis.). Protein-binding 96-well plates were from Millipore (Bedford, Mass.). Salts and buffering agents were from SIGMA.RTM. (St. Louis, Mo.). MALDI matrices were from ALDRICH.RTM. (Milwaukee, Wis.).

Protein Purification

[0284] Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle 3 was purified from expression media using a C-terminal hexa-histidine tag. The robotic purification is an adaptation of the protocol provided by Novagen for their HisBind resin. Briefly, a 150 uL (.mu.L) settled volume of resin is poured into the wells of a 96-well lysate-binding plate, washed with 3 volumes of water and charged with 5 volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media is diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) and loaded onto the columns. After draining, the columns are washed with 10 volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9). The eluted glycoproteins are evaporated to dryness by lyophilyzation.

Release of N-Linked Glycans

[0285] The glycans are released and separated from the glycoproteins by a modification of a previously reported method (Papac, et al. A. J. S. (1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) are wetted with 100 uL of methanol, washed with 3.times.150 uL of water and 50 uL of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum after each addition. The dried protein samples are dissolved in 30 uL of RCM buffer and transferred to the wells containing 10 uL of RCM buffer. The wells are drained and washed twice with RCM buffer. The proteins are reduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37.degree. C. The wells are washed three times with 300 uL of water and carboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30 min in the dark at room temperature. The wells are again washed three times with water and the membranes blocked by the addition of 100 uL of 1% PVP 360 in water for 1 hr at room temperature. The wells are drained and washed three times with 300 uL of water and deglycosylated by the addition of 30 uL of 10 mM NH4HCO3 pH 8.3 containing one milliunit of N-glycanase (Glyko). After 16 hours at 37.degree. C., the solution containing the glycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

[0286] Molecular weights of the glycans were determined using a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer using delayed extraction. The dried glycans from each well were dissolved in 15 uL of water and 0.5 uL spotted on stainless steel sample plates and mixed with 0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowed to dry.

[0287] Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) with a 4 ns pulse time. The instrument was operated in the delayed extraction mode with a 125 ns delay and an accelerating voltage of 20 kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, the internal pressure was less than 5.times.10-7 torr, and the low mass gate was 875 Da. Spectra were generated from the sum of 100-200 laser pulses and acquired with a 2 GHz digitizer. Man5 oligosaccharide was used as an external molecular weight standard. All spectra were generated with the instrument in the positive ion mode. The estimated mass accuracy of the spectra was 0.5%.

Materials:

[0288] MOPS, sodium cacodylate, manganese chloride, UDP-galactose and CMP-N-acetylneuraminic acid were from SIGMA.RTM., Saint Louis, Mo. Trifluroacetic acid (TFA) was from SIGMA/ALDRICH.RTM., Saint Louis, Mo. Recombinant rat alpha-2,6-sialyltransferase from Spodoptera frugiperda and beta-1,4-galactosyltransferase from bovine milk were from CALBIOCHEM.RTM., San Diego, Calif.

.beta.-N-Acetylhexosaminidase Digestion

[0289] The glycans were released and separated from the glycoproteins by a modification of a previously reported method (Papac, et al. A. J. S. (1998) Glycobiology 8, 445-454). After the proteins were reduced and carboxymethylated, and the membranes blocked, the wells were washed three time with water. The protein was deglycosylated by the addition of 30 .mu.l of 10 mM NH.sub.4HCO.sub.3 pH 8.3 containing one milliunit of N-glycanase (Glyko, Novato, Calif.). After 16 hr at 37.degree. C., the solution containing the glycans was removed by centrifugation and evaporated to dryness. The glycans were then dried in SC210A speed vac (Thermo Savant, Halbrook, N.Y.). The dried glycans were put in 50 mM NH.sub.4Ac pH 5.0 at 37.degree. C. overnight and 1 mU of hexos (Glyko, Novato, Calif.) was added.

Galactosyltransferase Reaction

[0290] Approximately 2 mg of protein (r-K3:hPg [PBP6-5]) was purified by nickel-affinity chromatography, extensively dialyzed against 0.1% TFA, and lyophilized to dryness. The protein was redissolved in 150 .mu.L of 50 mM MOPS, 20 mM MnCl2, pH7.4. After addition of 32.5 .mu.g (533 nmol) of UDP-galactose and 4 mU of .beta.1,4-galactosyltransferase, the sample was incubated at 37.degree. C. for 18 hours. The samples were then dialyzed against 0.1% TFA for analysis by MALDI-TOF mass spectrometry.

[0291] The spectrum of the protein reacted with galactosyltransferase showed an increase in mass consistent with the addition of two galactose moieties when compared with the spectrum of a similar protein sample incubated without enzyme. Protein samples were next reduced, carboxymethylated and deglycosylated with PNGase F. The recovered N-glycans were analyzed by MALDI-TOF mass spectrometry. The mass of the predominant glycan from the galactosyltransferase reacted protein was greater than that of the control glycan by a mass consistent with the addition of two galactose moieties (325.4 Da).

Sialyltransferase Reaction

[0292] After resuspending the (galactosyltransferase reacted) proteins in 10 .mu.L of 50 mM sodium cacodylate buffer pH6.0, 300 .mu.g (488 nmol) of CMP-N-acetylneuraminic acid (CMP-NANA) dissolved in 15 .mu.L of the same buffer, and 5 .mu.L (2 mU) of recombinant .alpha.-2,6 sialyltransferase were added. After incubation at 37.degree. C. for 15 hours, an additional 200 .mu.g of CMP-NANA and 1 mU of sialyltransferase were added. The protein samples were incubated for an additional 8 hours and then dialyzed and analyzed by MALDI-TOF-MS as above.

[0293] The spectrum of the glycoprotein reacted with sialyltransferase showed an increase in mass when compared with that of the starting material (the protein after galactosyltransferase reaction). The N-glycans were released and analyzed as above. The increase in mass of the two ion-adducts of the predominant glycan was consistent with the addition of two sialic acid residues (580 and 583 Da).

EXAMPLE 3

Identification, Cloning and Deletion of the ALG9 and ALG 12 Genes in P. pastoris

[0294] Similar to Example 1, the ALG9p and ALG12 sequences, respectively from S. cerevisiae, Drosophila melanogaster, Homo sapiens, etc., is aligned and regions of high homology are used to design degenerate primers. These primers are employed in a PCR reaction on genomic DNA from the P. pastoris. The resulting initial PCR product is subcloned, sequenced and used to probe a Southern blot of genomic DNA from P. pastoris with high stringency (Sambrook et al., 1989). Hybridization is observed. This Southern blot is used to map the genomic loci. Genomic fragments are cloned by digesting genomic DNA and ligating those fragments in the appropriate size-range into pUC19 to create a P. pastoris subgenomic library. This subgenomic library is transformed into E. coli and several hundred clones tested by colony PCR, using primers designed based on the sequence of the initial PCR product. The clones containing the predicted genes are sequenced and open reading frames identified. Primers for construction of an alg9::NAT.sup.R deletion allele, using a PCR overlap method (Davidson et al., 2002), are designed. The resulting deletion allele is transformed into two P. pastoris strains and NAT resistant colonies are selected. These colonies are screened by PCR and transformants obtained in which the ALG9 ORF is replaced with the och1::NAT.sup.R mutant allele. See generally, Cipollo et al. Glycobiology 2002 (12)11:749-762; Chantret et al. J. Biol. Chem. Jul. 12, 2002 (277)28:25815-25822; Cipollo et al. J. Biol. Chem. Feb. 11, 2000 (275)6:4267-4277; Burda et al. Proc. Natl. Acad. Sci. U.S.A. July 1996 (93):7160-7165; Karaoglu et al. Biochemistry 2001, 40, 12193-12206; Grimme et al. J. Biol. Chem. July 20, 2001 (276)29:27731-27739; Verostek et al. J. Biol. Chem. Jun. 5, 1993 (268)16:12095-12103; Huffaker et al. Proc. Natl. Acad. Sci. U.S.A. December 1983 (80):7466-7470.

EXAMPLE 4

Identification, Cloning and Expression of Alpha 1,2-3 Mannosidase From Xanthomonas Manihotis

[0295] The alpha 1,2-3 Mannosidase from Xanthomonas Manihotis has two activities: an alpha-1,2 and an alpha-1,3 mannosidase. The methods of the invention may also use two independent mannosidases having these activities, which may be similarly identified and cloned from a selected organism of interest.

[0296] As described by Landry et al., alpha-mannosidases can be purified from Xanthomonas sp., such as Xanthomonas manihotis. X. manihotis can be purchased from the American Type Culture Collection (ATCC catalog number 49764) (Xanthomonas axonopodis Starr and Garces pathovar manihotis deposited as Xanthomonas manihotis (Arthaud-Berthet) Starr). Enzymes are purified from crude cell-extracts as previously described (Wong-Madden, S. T. and Landry, D. (1995) Purification and characterization of novel glycosidases from the bacterial genus Xanthomonas; and Landry, D. U.S. Pat. No. 6,300,113 B1 Isolation and composition of novel Glycosidases). After purification of the mannosidase, one of several methods are used to obtain peptide sequence tags (see, e.g., W. Quadroni M et al. (2000). A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification. Anal Chem (2000) March 1; 72(5):1006-14; Wilkins M R et al. Rapid protein identification using N-terminal "sequence tag" and amino acid analysis. Biochem Biophys Res Commun (1996) April 25; 221(3):609-13; and Tsugita A. (1987) Developments in protein microsequencing. Adv Biophys (1987) 23:81-113).

[0297] Sequence tags generated using a method above are then used to generate sets of degenerate primers using methods well-known to the skilled worker. Degenerate primers are used to prime DNA amplification in polymerase chain reactions (e.g., using Taq polymerase kits according to manufacturers' instructions) to amplify DNA fragments. The amplified DNA fragments are used as probes to isolate DNA molecules comprising the gene encoding a desired mannosidase, e.g., using standard Southern DNA hybridization techniques to identify and isolate (clone) genomic pieces encoding the enzyme of interest. The genomic DNA molecules are sequenced and putative open reading frames and coding sequences are identified. A suitable expression construct encoding for the glycosidase of interest can then be generated using methods described herein and well-known in the art.

[0298] Nucleic acid fragments comprising sequences encoding alpha 1,2-3 mannosidase activity (or catalytically active fragments thereof) are cloned into appropriate expression vectors for expression, and preferably targeted expression, of these activities in an appropriate host cell according to the methods set forth herein.

EXAMPLE 5

Identification, Cloning and Expression of the ALG6 Gene in P. pastoris

[0299] Similar to Example 1, the ALG6p sequences from S. cerevisiae, Drosophila melanogaster, Homo sapiens etc., are aligned and regions of high homology are used to design degenerate primers. These primers are employed in a PCR reaction on genomic DNA from the P. pastoris. The resulting initial PCR product is subcloned, sequenced and used to probe a Southern blot of genomic DNA from P. pastoris with high stringency (Sambrook et al, 1989). Hybridization is observed. This Southern blot is used to map the genomic loci. Genomic fragments are cloned by digesting genomic DNA and ligating those fragments in the appropriate size-range into pUC19 to create a P. pastoris subgenomic library. This subgenomic library is transformed into E. coli and several hundred clones are tested by colony PCR, using primers designed based on the sequence of the initial PCR product. The clones containing the predicted genes are sequenced and open reading frames identified. Primers for construction of an alg6::NAT.sup.R deletion allele, using a PCR overlap method (Davidson et al, 2002), are designed and the resulting deletion allele is transformed into two P. pastoris strains and NAT resistant colonies selected. These colonies are screened by PCR and transformants are obtained in which the ALG6 ORF is replaced with the och1::NAT.sup.R mutant allele. See, e.g., Imbach et al. Proc. Natl. Acad. Sci. U.S.A. June 1999 (96)6982-6987.

[0300] Nucleic acid fragments comprising sequences encoding Alg6p (or catalytically active fragments thereof) are cloned into appropriate expression vectors for expression, and preferably targeted expression, of these activities in an appropriate host cell according to the methods set forth herein. The cloned ALG6 gene can be brought under the control of any suitable promoter to achieve overexpression. Even expression of the gene under the control of its own promoter is possible. Expression from multicopy plasmids will generate high levels of expression ("overexpression").

EXAMPLE 6

Cloning and Expression Of GnT III to Produce Bisecting GlcNAcs which Boost Antibody Functionality

A. Background

[0301] The addition of an N-acetylglucosamine to the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 structure by N-acetylglucosaminyltransferases III yields a so-called bisected N-glycan (see FIG. 3). This structure has been implicated in greater antibody-dependent cellular cytotoxicity (ADCC) (Umana et al. 1999).

[0302] A host cell such as a yeast strain capable of producing glycoproteins with bisected N-glycans is engineered according to the invention, by introducing into the host cell a GnTIII activity. Preferably, the host cell is transformed with a nucleic acid that encodes GnTIII (e.g., a mammalian such as the murine GnT III shown in FIG. 32) or a domain thereof having enzymatic activity, optionally fused to a heterologous cell signal targeting peptide (e.g., using the libraries and associated methods of the invention.)

[0303] IgGs consist of two heavy-chains (V.sub.H, C.sub.H1, C.sub.H2 and C.sub.H3 in FIG. 30), interconnected in the hinge region through three disulfide bridges, and two light chains (V.sub.L, C.sub.L in FIG. 30). The light chains (domains V.sub.L and C.sub.L) are linked by another disulfide bridge to the C.sub.H1 portion of the heavy chain and together with the C.sub.H1 and V.sub.H fragment make up the so-called Fab region. Antigens bind to the terminal portion of the Fab region. The Fc region of IgGs consists of the C.sub.H3, the C.sub.H2 and the hinge region and is responsible for the exertion of so-called effector functions (see below).

[0304] The primary function of antibodies is binding to an antigen. However, unless binding to the antigen directly inactivates the antigen (such as in the case of bacterial toxins), mere binding is meaningless unless so-called effector-functions are triggered. Antibodies of the IgG subclass exert two major effector-functions: the activation of the complement system and induction of phagocytosis. The complement system consists of a complex group of serum proteins involved in controlling inflammatory events, in the activation of phagocytes and in the lytical destruction of cell membranes. Complement activation starts with binding of the C1 complex to the Fc portion of two IgGs in close proximity. C1 consists of one molecule, C1q, and two molecules, C1r and C1s. Phagocytosis is initiated through an interaction between the IgG's Fc fragment and Fc-gamma-receptors (FcyRI, II and III in FIG. 30). Fc receptors are primarily expressed on the surface of effector cells of the immune system, in particular macrophages, monocytes, myeloid cells and dendritic cells.

[0305] The C.sub.H2 portion harbors a conserved N-glycosylation site at asparagine 297 (Asp297). The Asp297 N-glycans are highly heterogeneous and are known to affect Fc receptor binding and complement activation. Only a minority (i.e., about 15-20%) of IgGs bears a disialylated, and 3-10% have a monosialylated N-glycan (reviewed in Jefferis, R., Glycosylation of human IgG Antibodies. BioPharm, 2001). Interestingly, the minimal N-glycan structure shown to be necessary for fully functional antibodies capable of complement activation and Fc receptor binding is a pentasacharide with terminal N-acetylglucosamine residues (GlcNAc.sub.2Man.sub.3) (reviewed in Jefferis, R., Glycosylation of human IgG Antibodies. BioPharm, 2001). Antibodies with less than a GlcNAc.sub.2Man.sub.3 N-glycan or no N-glycosylation at Asp297 might still be able to bind an antigen but most likely will not activate the crucial downstream events such as phagocytosis and complement activation. In addition, antibodies with fungal-type N-glycans attached to Asp297 will in all likelihood solicit an immune-response in a mammalian organism which will render that antibody useless as a therapeutic glycoprotein.

B. Cloning and Expression of GnTIII

[0306] The DNA fragment encoding part of the mouse GnTIII protein lacking the TM domain is PCR amplified from murine (or other mammalian) genomic DNA using forward 5'-TCCTGGCGCGCCTTCCCGAGAGAACTGGCCTCCCTC-3'(SEQ ID NO. 15) and

[0307] 5'-AATTAATTAACCCTAGCCCTCCGCTGTATCCAACTTG-3' (SEQ ID NO. 16) reversed primers. Those primers include AscI and PacI restriction sites that will be used for cloning into the vector suitable for the fusion with leader library. The nucleic acid and amino acid sequence of murine GnTIII is shown in FIG. 32.

C. Cloning of Immunoulobulin Encoding Sequences

[0308] Protocols for the cloning of the variable regions of antibodies, including primer sequences, have been published previously. Sources of antibodies and encoding genes can be, among others, in vitro immunized human B cells (see, e.g., Borreback, C. A. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 3995-3999), periphal blood lymphocytes or single human B cells (see, e.g., Lagerkvist, A. C. et al. (1995) Biotechniques 18, 862-869; and Terness, P. et al. (1997) Hum. Immunol. 56, 17-27) and transgenic mice containing human immunoglobulin loci, allowing the creation of hybridoma cell-lines.

[0309] Using standard recombinant DNA techniques, antibody-encoding nucleic acid sequences can be cloned. Sources for the genetic information encoding immunoglobulins of interest are typically total RNA preparations from cells of interest, such as blood lymphocytes or hybridoma cell lines. For example, by employing a PCR based protocol with specific primers, variable regions can be cloned via reverse transcription initiated from a sequence-specific primer hybridizing to the IgG C.sub.H1 domain site and a second primer encoding amino acids 111-118 of the murine kappa constant region. The V.sub.H and V.sub.K encoding cDNAs will then be amplified as previously published (see, e.g., Graziano, R. F. et al. (1995) J Immunol. 155(10): p. 4996-5002; Welschof, M. et al. (1995) J. Immunol. Methods 179, 203-214; and Orlandi, R. et al. (1988) Proc. Natl. Acad. Sci. USA 86: 3833). Cloning procedures for whole immunoglobulins (heavy and light chains have also been published (see, e.g., Buckel, P. et al. (1987) Gene 51:13-19; Recinos A 3.sup.rd et al. (1994) Gene 149: 385-386; (1995) Gene June 9; 158(2):311-2; and Recinos A 3.sup.rd et al. (1994) Gene November 18; 149(2):385-6). Additional protocols for the cloning and generation of antibody fragment and antibody expression constructs have been described in Antibody Engineering, R. Kontermann and S. Dubel (2001), Editors, Springer Verlag: Berlin Heidelberg New York.

[0310] Fungal expression plasmids encoding heavy and light chain of immunoglobulins have been described (see, e.g., Abdel-Salam, H. A. et al. (2001) Appl. Microbiol. Biotechnol. 56: 157-164; and Ogunjimi, A. A. et al. (1999) Biotechnology Letters 21: 561-567). One can thus generate expression plasmids harboring the constant regions of immunoglobulins. To facilitate the cloning of variable regions into these expression vectors, suitable restriction sites can be placed in close proximity to the termini of the variable regions. The constant regions can be constructed in such a way that the variable regions can be easily in-frame fused to them by a simple restriction-digest/ligation experiment. FIG. 31 shows a schematic overview of such an expression construct, designed in a very modular way, allowing easy exchange of promoters, transcriptional terminators, integration targeting domains and even selection markers.

[0311] As shown in FIG. 31, V.sub.L as well as V.sub.H domains of choice can be easily cloned in-frame with C.sub.L and the C.sub.H regions, respectively. Initial integration is targeted to the P. pastoris AOX locus (or homologous locus in another fungal cell) and the methanol-inducible AOX promoter will drive expression. Alternatively, any other desired constitutive or inducible promoter cassette may be used. Thus, if desired, the 5'AOX and 3'AOX regions as well as transcriptional terminator (TT) fragments can be easily replaced with different TT, promoter and integration targeting domains to optimize expression. Initially the alpha-factor secretion signal with the standard KEX protease site is employed to facilitate secretion of heavy and light chains. The properties of the expression vector may be further refined using standard techniques.

[0312] An Ig expression vector such as the one described above is introduced into a host cell of the invention that expresses GnTIII, preferably in the Golgi apparatus of the host cell. The Ig molecules expressed in such a host cell comprise N-glycans having bisecting GlcNAcs.

EXAMPLE 7

Cloning and Expression of GnT-IV (UDP-GlcNAc:alpha-1,3-D-mannoside beta-1,4-N-Acetylglucosaminyltransferase IV) and GnT-V (beta 1-6-N-acetylglucosaminyltransferase)

[0313] GnTIV-encoding cDNAs were isolated from bovine and human cells (Minowa, M. T. et al. (1998) J. Biol. Chem. 273 (19), 11556-11562; and Yoshida, A. et al. (1999) Glycobiology 9 (3), 303-310. The DNA fragments encoding full length and a part of the human GnT-IV protein (FIG. 33) lacking the TM domain are PCR amplified from the cDNA library using forward [0314] 5'-AATGAGATGAGGCTCCGCAATGGAACTG-3' (SEQ ID NO. 17), [0315] 5'-CTGATTGCTTATCAACGAGAATTCCTTG-3' (SEQ ID NO. 18), and reverse [0316] 5'-TGTTGGTTTCTCAGATGATCAGTTGGTG-3'(SEQ ID NO. 19) primers, respectively. The resulting PCR products are cloned and sequenced.

[0317] Similarly, genes encoding GnT-V protein have been isolated from several mammalian species, including mouse. (See, e.g., Alverez, K. et al. Glycobiology 12 (7), 389-394 (2002)). The DNA fragments encoding full length and a part of the mouse GnT-V protein (FIG. 34) lacking the TM domain are PCR amplified from the cDNA library using forward 5'-AGAGAGAGATGGCTTTCTTTTCTCCCTGG-3' (SEQ ID NO. 20), 5'-AAATCAAGTGGATGAAGGACATGTGGC-3' (SEQ ID NO. 21), and reverse 5'-AGCGATGCTATAGGCAGTCTTTGCAGAG-3' (SEQ ID NO. 22) primers, respectively. The resulting PCR products are cloned and sequenced.

[0318] Nucleic acid fragments comprising sequences encoding GnT IV or V (or catalytically active fragments thereof) are cloned into appropriate expression vectors for expression, and preferably targeted expression, of these activities in an appropriate host cell according to the methods set forth herein.

REFERENCES

[0319] Aebi, M., J. Gassenhuber, et al. (1996). "Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae." Glycobiology 6(4): 439-444. [0320] Altmann, F., E. Staudacher, et al. (1999). "Insect cells as hosts for the expression of recombinant glycoproteins." Glycoconjugate Journal 16(2): 109-123. [0321] Andersen, D. C. and C. F. Goochee (1994). "The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins." Current Opinion in Biotechnology 5: 546-549. [0322] Bardor, M., L. Faye, et al. (1999). "Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants." Trends in Plant Science 4(9): 376-380. [0323] Bretthauer, R. K. and F. J. Castellino (1999). "Glycosylation of Pichia pastoris-derived proteins." Biotechnology and Applied Biochemistry 30: 193-200. [0324] Burda, P. and M. Aebi (1999). "The dolichol pathway of N-linked glycosylation." Biochimica Et Biophysica Acta-General Subjects 1426(2): 239-257. 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Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 80(24): 7466-7470. [0330] Jarvis, D. L., Z. S. Kawar, et al. (1998). "Engineering N-glycosylation pathways in the baculovirus-insect cell system." Current Opinion in Biotechnology 9(5): 528-533. [0331] Kimura, T., N. Kitamoto, et al. (1997). "A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis." Molecular & General Genetics 254(2): 139-147. [0332] Kimura, T., T. Komiyama, et al. (1999). "N-glycosylation is involved in the sensitivity of Saccharomyces cerevisiae to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895." Applied Microbiology and Biotechnology 51(2): 176-184. [0333] Malissard, M., S. Zeng, et al. (2000). "Expression of functional soluble forms of human beta-1,4- galactosyltransferase I, alpha-2,6-sialyltransferase, and alpha-1,3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris." Biochemical and Biophysical Research Communications 267(1): 169-173. [0334] Maras, M. and R. Contreras (1994). Methods of Modifying Carbohydrate Moieties. United States, Alko Group Ltd., Helsinki, Finland. [0335] Maras, M., A. De Bruyn, et al. (1999). "In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei." Febs Letters 452(3): 365-370. [0336] Maras, M., X. Saelens, et al. (1997). "In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides--Evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei." European Journal of Biochemistry 249(3): 701-707. [0337] Martinet, W., M. Maras, et al. (1998). "Modification of the protein glycosylation pathway in the methylotrophic yeast Pichia pastoris." Biotechnology Letters 20(12): 1171-1177. [0338] McGarvey, P. B., J. Hammond, et al. (1995). "Expression of the Rabies Virus Glycoprotein in Transgenic Tomatoes." Bio-Technology 13(13): 1484-1487. [0339] Moens, S. and J. Vanderleyden (1997). "Glycoproteins in prokaryotes." Archives of Microbiology 168(3): 169-175. [0340] Nakanishishindo, Y., K. Nakayama, et al. (1993). "Structure of the N-Linked Oligosaccharides That Show the Complete Loss of Alpha-1,6-Polymannose Outer Chain From Och1, Och1 Mnn1, and Och1 Mnn1 Alg3 Mutants of Saccharomyces-Cerevisiae." Journal of Biological Chemistry 268(35): 26338-26345. [0341] Raju, T. S., J. B. Briggs, et al. (2000). "Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics." Glycobiology 10(5): 477-486. [0342] Sharma, C. B., R. Knauer, et al. (2001). "Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the DoI-P-Man: Man(5)GlcNAc(2)-PP-DoI mannosyltransferase." Biological Chemistry 382(2): 321-328. [0343] Staub, J. M., B. Garcia, et al. (2000). "High-yield production of a human therapeutic protein in tobacco chloroplasts." Nature Biotechnology 18(3): 333-338. [0344] Takeuchi, M. (1997). "Trial for molecular breeding of yeast for the production of glycoprotein therapeutics." Trends in Glycoscience and Glycotechnology 9: S29-S35. [0345] Umana et al., Nat Biotechnol. 1999a February (17)176-180. (Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibodydependent cellular cytotoxic activity) [0346] Umana et al., Biotechnol Bioeng. 1999b December 5; 65(5):542-549. (Regulated Overexpression of glycosyltransferase). [0347] Verostek, M. F., P. H. Atkinson, et al. (1993). "Glycoprotein-Biosynthesis in the Alg3 Saccharomyces-Cerevisiae Mutant 0.1. Role of Glucose in the Initial Glycosylation of Invertase in the Endoplasmic-Reticulum." Journal of Biological Chemistry 268(16): 12095-12103. [0348] Verostek, M. F., P. H. Atkinson, et al. (1993). "Glycoprotein-Biosynthesis in the Alg3 Saccharomyces-Cerevisiae Mutant 0.2. Structure of Novel Man6-10glcnac2 Processing Intermediates On Secreted Invertase." Journal of Biological Chemistry 268(16): 12104-12115. [0349] Weikert, S., D. Papac, et al. (1999). "Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins." Nature Biotechnology 17(11): 1116-1121. [0350] Werner, R. G., W. Noe, et al. (1998). "Appropriate mammalian expression systems for biopharmaceuticals." Arzneimittel-Forschung-Drug Research 48(8): 870-880. [0351] Yang, M. and M. Butler (2000). "Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation." Biotechnology and Bioengineering 68(4): 370-380.Zufferey, R., R. Knauer, et al. (1995). "Stt3, a Highly Conserved Protein Required for Yeast Oligosaccharyl Transferase-Activity in-Vivo." EMBO Journal 14(20): 4949-4960. 45

Sequence CWU 1

1

58135DNAArtificial SequenceDescription of Artificial Sequence Primer 1ggtgttttgt tttctagatc tttgcaytay cartt 35236DNAArtificial SequenceDescription of Artificial Sequence Primer 2agaatttggt gggtaagaat tccarcacca ytcrtg 36332DNAArtificial SequenceDescription of Artificial Sequence Primer 3cctaagctgg tatgcgttct ctttgccata tc 32430DNAArtificial SequenceDescription of Artificial Sequence Primer 4gcggcataaa caataataga tgctataaag 30520DNAArtificial SequenceDescription of Artificial Sequence Primer 5aattaaccct cactaaaggg 20622DNAArtificial SequenceDescription of Artificial Sequence Primer 6gtaatacgac tcactatagg gc 22724DNAArtificial SequenceDescription of Artificial Sequence Primer 7ccacatcatc cgtgctacat atag 24844DNAArtificial SequenceDescription of Artificial Sequence Primer 8acgaggcaag ctaaacagat ctcgaagtat cgagggttat ccag 44944DNAArtificial SequenceDescription of Artificial Sequence Primer 9ccatccagtg tcgaaaacga gccaatggtt catgtctata aatc 441024DNAArtificial SequenceDescription of Artificial Sequence Primer 10agcctcagcg ccaacaagcg atgg 241144DNAArtificial SequenceDescription of Artificial Sequence Primer 11ctggataacc ctcgatactt cgagatctgt ttagcttgcc tcgt 441244DNAArtificial SequenceDescription of Artificial Sequence Primer 12gatttataga catgaaccat tggctcgttt tcgacactgg atgg 441320DNAArtificial SequenceDescription of Artificial Sequence Primer 13atcctttacc gatgctgtat 201427DNAArtificial SequenceDescription of Artificial Sequence Primer 14ataacagtat gtgttacacg cgtgtag 271536DNAArtificial SequenceDescription of Artificial Sequence Primer 15tcctggcgcg ccttcccgag agaactggcc tccctc 361637DNAArtificial SequenceDescription of Artificial Sequence Primer 16aattaattaa ccctagccct ccgctgtatc caacttg 371728DNAArtificial SequenceDescription of Artificial Sequence Primer 17aatgagatga ggctccgcaa tggaactg 281828DNAArtificial SequenceDescription of Artificial Sequence Primer 18ctgattgctt atcaacgaga attccttg 281928DNAArtificial SequenceDescription of Artificial Sequence Primer 19tgttggtttc tcagatgatc agttggtg 282029DNAArtificial SequenceDescription of Artificial Sequence Primer 20agagagagat ggctttcttt tctccctgg 292127DNAArtificial SequenceDescription of Artificial Sequence Primer 21aaatcaagtg gatgaaggac atgtggc 272228DNAArtificial SequenceDescription of Artificial Sequence Primer 22agcgatgcta taggcagtct ttgcagag 28234PRTSaccharomyces cerevisiae 23His Asp Glu Leu 1 24458PRTSaccharomyces cerevisiaeMOD_RES(304)..(318)Variable amino acid 24Met Glu Gly Glu Gln Ser Pro Gln Gly Glu Lys Ser Leu Gln Arg Lys 1 5 10 15 Gln Phe Val Arg Pro Pro Leu Asp Leu Trp Gln Asp Leu Lys Asp Gly 20 25 30 Val Arg Tyr Val Ile Phe Asp Cys Arg Ala Asn Leu Ile Val Met Pro 35 40 45 Leu Leu Ile Leu Phe Glu Ser Met Leu Cys Lys Ile Ile Ile Lys Lys 50 55 60 Val Ala Tyr Thr Glu Ile Asp Tyr Lys Ala Tyr Met Glu Gln Ile Glu 65 70 75 80 Met Ile Gln Leu Asp Gly Met Leu Asp Tyr Ser Gln Val Ser Gly Gly 85 90 95 Thr Gly Pro Leu Val Tyr Pro Ala Gly His Val Leu Ile Tyr Lys Met 100 105 110 Met Tyr Trp Leu Thr Glu Gly Met Asp His Val Glu Arg Gly Gln Val 115 120 125 Phe Phe Arg Tyr Leu Tyr Leu Leu Thr Leu Ala Leu Gln Met Ala Cys 130 135 140 Tyr Tyr Leu Leu His Leu Pro Pro Trp Cys Val Val Leu Ala Cys Leu 145 150 155 160 Ser Lys Arg Leu His Ser Ile Tyr Val Leu Arg Leu Phe Asn Asp Cys 165 170 175 Phe Thr Thr Leu Phe Met Val Val Thr Val Leu Gly Ala Ile Val Ala 180 185 190 Ser Arg Cys His Gln Arg Pro Lys Leu Lys Lys Ser Leu Ala Leu Val 195 200 205 Ile Ser Ala Thr Tyr Ser Met Ala Val Ser Ile Lys Met Asn Ala Leu 210 215 220 Leu Tyr Phe Pro Ala Met Met Ile Ser Leu Phe Ile Leu Asn Asp Ala 225 230 235 240 Asn Val Ile Leu Thr Leu Leu Asp Leu Val Ala Met Ile Ala Trp Gln 245 250 255 Val Ala Val Ala Val Pro Phe Leu Arg Ser Phe Pro Gln Gln Tyr Leu 260 265 270 His Cys Ala Phe Asn Phe Gly Arg Lys Phe Met Tyr Gln Trp Ser Ile 275 280 285 Asn Trp Gln Met Met Asp Glu Glu Ala Phe Asn Asp Lys Arg Phe Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Val 305 310 315 320 Thr Arg Tyr Pro Arg Ile Leu Pro Asp Leu Trp Ser Ser Leu Cys His 325 330 335 Pro Leu Arg Lys Asn Ala Val Leu Asn Ala Asn Pro Ala Lys Thr Ile 340 345 350 Pro Phe Val Leu Ile Ala Ser Asn Phe Ile Gly Val Leu Phe Ser Arg 355 360 365 Ser Leu His Tyr Gln Phe Leu Ser Trp Tyr His Trp Thr Leu Pro Ile 370 375 380 Leu Ile Phe Trp Ser Gly Met Pro Phe Phe Val Gly Pro Ile Trp Tyr 385 390 395 400 Val Leu His Glu Trp Cys Trp Asn Ser Tyr Pro Pro Asn Ser Gln Xaa 405 410 415 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Ser Gly Ser Val Ala Leu Ala Lys Ser His Leu Arg 435 440 445 Thr Thr Ser Ser Met Glu Lys Lys Leu Asn 450 455 25443PRTSaccharomyces cerevisiaeMOD_RES(333)..(347)Variable amino acid 25Trp Gln Asp Leu Lys Asp Gly Val Arg Tyr Val Ile Phe Asp Cys Arg 1 5 10 15 Ala Asn Leu Ile Val Met Pro Leu Leu Ile Leu Phe Glu Ser Met Leu 20 25 30 Cys Lys Ile Ile Ile Lys Lys Val Ala Tyr Thr Glu Ile Asp Tyr Lys 35 40 45 Ala Tyr Met Glu Gln Ile Glu Met Ile Gln Leu Asp Gly Met Leu Asp 50 55 60 Tyr Ser Gln Val Ser Gly Gly Thr Gly Pro Leu Val Tyr Pro Ala Gly 65 70 75 80 His Val Leu Ile Tyr Lys Met Met Tyr Trp Leu Thr Glu Gly Met Asp 85 90 95 His Val Glu Arg Gly Gln Val Phe Phe Arg Tyr Leu Tyr Leu Leu Thr 100 105 110 Leu Ala Leu Gln Met Ala Cys Tyr Tyr Leu Leu His Leu Pro Pro Trp 115 120 125 Cys Val Val Leu Ala Cys Leu Ser Lys Arg Leu His Ser Ile Tyr Val 130 135 140 Leu Arg Leu Phe Asn Asp Cys Phe Thr Thr Leu Phe Met Val Val Thr 145 150 155 160 Val Leu Gly Ala Ile Val Ala Ser Arg Cys His Gln Arg Pro Lys Leu 165 170 175 Lys Lys His Gln Thr Cys Lys Val Pro Pro Phe Val Phe Phe Phe Met 180 185 190 Cys Cys Ala Ser Tyr Arg Val His Ser Ile Phe Val Leu Arg Leu Phe 195 200 205 Asn Asp Pro Val Ala Met Val Leu Leu Phe Leu Ser Ile Asn Leu Leu 210 215 220 Leu Ala Gln Arg Trp Gly Trp Gly Ser Leu Ala Leu Val Ile Ser Ala 225 230 235 240 Thr Tyr Ser Met Ala Val Ser Ile Lys Met Asn Ala Leu Leu Tyr Phe 245 250 255 Pro Ala Met Met Ile Ser Leu Phe Ile Leu Asn Asp Ala Asn Val Ile 260 265 270 Leu Thr Leu Leu Asp Leu Val Ala Met Ile Ala Trp Gln Val Ala Val 275 280 285 Ala Val Pro Phe Leu Arg Ser Phe Pro Gln Gln Tyr Leu His Cys Ala 290 295 300 Phe Asn Phe Gly Arg Lys Phe Met Tyr Gln Trp Ser Ile Asn Trp Gln 305 310 315 320 Met Met Asp Glu Glu Ala Phe Asn Asp Lys Arg Phe Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Val Thr Arg Tyr 340 345 350 Pro Arg Ile Leu Pro Asp Leu Trp Ser Ser Leu Cys His Pro Leu Arg 355 360 365 Lys Asn Ala Val Leu Asn Ala Asn Pro Ala Lys Thr Ile Pro Phe Val 370 375 380 Leu Ile Ala Ser Asn Phe Ile Gly Val Leu Phe Ser Arg Ser Leu His 385 390 395 400 Tyr Gln Phe Leu Ser Trp Tyr His Trp Thr Leu Pro Ile Leu Ile Phe 405 410 415 Trp Ser Gly Met Pro Phe Phe Val Gly Pro Ile Trp Tyr Val Leu His 420 425 430 Glu Trp Cys Trp Asn Ser Tyr Pro Pro Asn Ser 435 440 261377DNASaccharomyces cerevisiae 26atggaaggtg aacagtctcc gcaaggtgaa aagtctctgc aaaggaagca atttgtcaga 60cctccgctgg atctgtggca ggatctcaag gacggtgtgc gctacgtgat cttcgattgt 120agggccaatc ttatcgttat gccccttttg attttgttcg aaagcatgct gtgcaagatt 180atcattaaga aggtagctta cacagagatc gattacaagg cgtacatgga gcagatcgag 240atgattcagc tcgatggcat gctggactac tctcaggtga gtggtggaac gggcccgctg 300gtgtatccag caggccacgt cttgatctac aagatgatgt actggctaac agagggaatg 360gaccacgttg agcgcgggca agtgtttttc agatacttgt atctccttac actggcgtta 420caaatggcgt gttactacct tttacatcta ccaccgtggt gtgtggtctt ggcgtgcctc 480tctaaaagat tgcactctat ttacgtgcta cggttattca atgattgctt cactactttg 540tttatggtcg tcacggtttt gggggctatc gtggccagca ggtgccatca gcgccccaaa 600ttaaagaagt cccttgcgct ggtgatctcc gcaacataca gtatggctgt gagcattaag 660atgaatgcgc tgttgtattt ccctgcaatg atgatttctc tattcatcct taatgacgcg 720aacgtaatcc ttactttgtt ggatctcgtt gcgatgattg catggcaagt cgcagttgca 780gtgcccttcc tgcgcagctt tccgcaacag tacctgcatt gcgcttttaa tttcggcagg 840aagtttatgt accaatggag tatcaattgg caaatgatgg atgaagaggc tttcaatgat 900aagaggttcc acttggccct tttaatcagc cacctgatag cgctcaccac actgttcgtc 960acaagatacc ctcgcatcct gcccgattta tggtcttccc tgtgccatcc gctgaggaaa 1020aatgcagtgc tcaatgccaa tcccgccaag actattccat tcgttctaat cgcatccaac 1080ttcatcggcg tcctattttc aaggtccctc cactaccagt ttctatcctg gtatcactgg 1140actttgccta tactgatctt ttggtcggga atgcccttct tcgttggtcc catttggtac 1200gtcttgcacg agtggtgctg gaattcctat ccaccaaact cacaagcaag cacgctattg 1260ttggcattga atactgttct gttgcttcta ttggccttga cgcagctatc tggttcggtc 1320gccctcgcca aaagccatct tcgtaccacc agctctatgg aaaaaaagct caactga 1377271395DNAPichia pastoris 27atgcctccga tagagccagc tgaaaggcca aagcttacgc tgaaaaatgt tatcggtgat 60ctagtggctc ttattcaaaa cgttttattt aacccagatt ttagtgtctt cgttgcacct 120cttttatggt tagctgattc cattgttatc aaggtgatca ttggcactgt ttcctacaca 180gatattgatt tttcttcata tatgcaacaa atctttaaaa ttcgacaagg agaattagat 240tatagcaaca tatttggtga caccggtcca ttggtttacc cagccggcca tgttcatgct 300tactcagtac tttcgtggta cagtgatggt ggagaagacg tcagtttcgt tcaacaagca 360tttggttggt tatacctagg ttgcttgtta ctatccatca gctcctactt tttctctggc 420ttagggaaaa tacctccggt ttattttgtt ttgttggtag cgtccaagag actgcattca 480atatttgtat tgagactctt caatgactgt ttaacaacat ttttgatgtt ggcaactata 540atcatccttc aacaagcaag tagctggagg aaagatggca caactattcc attatctgtc 600cctgatgctg cagatacgta cagtttagcc atctctgtaa agatgaatgc gctgctatac 660ctcccagcat tcctactact catatatctc atttgtgacg aaaatttgat taaagccttg 720gcacctgttc tagttttgat attggtgcaa gtaggagtcg gttattcgtt cattttaccg 780ttgcactatg atgatcaggc aaatgaaatt cgttctgcct actttagaca ggcttttgac 840tttagtcgcc aatttcttta taagtggacg gttaattggc gctttttgag ccaagaaact 900ttcaacaatg tccattttca ccagctcctg tttgctctcc atattattac gttagtcttg 960ttcatcctca agttcctctc tcctaaaaac attggaaaac cgcttggtag atttgtgttg 1020gacattttca aattttggaa gccaacctta tctccaacca atattatcaa cgacccagaa 1080agaagcccag attttgttta caccgtcatg gctactacca acttaatagg ggtgcttttt 1140gcaagatctt tacactacca gttcctaagc tggtatgcgt tctctttgcc atatctcctt 1200tacaaggctc gtctgaactt tatagcatct attattgttt atgccgctca cgagtattgc 1260tggttggttt tcccagctac agaacaaagt tccgcgttgt tggtatctat cttactactt 1320atcctgattc tcatttttac caacgaacag ttatttcctt ctcaatcggt ccctgcagaa 1380aaaaagaata cataa 139528418PRTPichia pastorisMOD_RES(209)..(223)Variable amino acid 28Arg Pro Lys Leu Thr Leu Lys Asn Val Ile Gly Asp Leu Val Ala Leu 1 5 10 15 Ile Gln Asn Val Leu Phe Asn Pro Asp Phe Ser Val Phe Val Ala Pro 20 25 30 Leu Leu Trp Leu Ala Asp Ser Ile Val Ile Lys Val Ile Ile Gly Thr 35 40 45 Val Ser Tyr Thr Asp Ile Asp Phe Ser Ser Tyr Met Gln Gln Ile Phe 50 55 60 Lys Ile Arg Gln Gly Glu Leu Asp Tyr Ser Asn Ile Phe Gly Asp Thr 65 70 75 80 Gly Pro Leu Val Tyr Pro Ala Gly His Val His Ala Tyr Ser Val Leu 85 90 95 Ser Trp Tyr Ser Asp Gly Gly Glu Asp Val Ser Phe Val Gln Gln Ala 100 105 110 Phe Gly Trp Leu Tyr Leu Gly Cys Leu Leu Leu Ser Ile Ser Ser Tyr 115 120 125 Phe Phe Ser Gly Leu Gly Lys Ile Pro Pro Val Tyr Phe Val Leu Leu 130 135 140 Val Ala Ser Lys Arg Leu His Ser Ile Phe Val Leu Arg Leu Phe Asn 145 150 155 160 Asp Cys Leu Thr Thr Phe Leu Met Leu Ala Thr Ile Ile Ile Leu Gln 165 170 175 Gln Ala Ser Ser Trp Arg Lys Asp Gly Thr Thr Ile Pro Leu Ser Val 180 185 190 Pro Asp Ala Ala Asp Thr Tyr Ser Leu Ala Ile Ser Val Lys Met Asn 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 210 215 220 Asp Glu Asn Leu Ile Lys Ala Leu Ala Pro Xaa Xaa Xaa Xaa Xaa Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa Tyr Ser Phe Ile Leu Pro Leu His Tyr Asp 245 250 255 Asp Gln Ala Asn Glu Ile Arg Ser Ala Tyr Phe Arg Gln Ala Phe Asp 260 265 270 Phe Ser Arg Gln Phe Leu Tyr Lys Trp Thr Val Asn Trp Arg Phe Leu 275 280 285 Ser Gln Glu Thr Phe Asn Asn Val His Phe His Gln Leu Leu Phe Ala 290 295 300 Leu His Ile Ile Thr Leu Val Leu Phe Ile Leu Lys Phe Leu Ser Pro 305 310 315 320 Lys Asn Ile Gly Lys Pro Leu Gly Arg Phe Val Leu Asp Ile Phe Lys 325 330 335 Phe Trp Lys Pro Thr Leu Ser Pro Thr Asn Ile Ile Asn Pro Asp Phe 340 345 350 Val Tyr Thr Val Met Ala Thr Thr Asn Leu Ile Gly Val Leu Phe Ala 355 360 365 Arg Ser Leu His Tyr Gln Phe Leu Ser Trp Tyr Ala Phe Ser Leu Pro 370 375 380 Tyr Leu Leu Tyr Lys Ala Arg Leu Asn Phe Ile Ala Ser Ile Ile Val 385 390 395 400 Tyr Ala Ala His Glu Tyr Cys Trp Leu Val Phe Pro Ala Thr Glu Gln 405 410 415 Ser Ser 29387PRTPichia pastorisMOD_RES(183)..(197)Variable amino acid 29Ser Val Phe Val Ala Pro Leu Leu Trp Leu Ala Asp Ser Ile Val Ile 1 5 10 15 Lys Val Ile Ile Gly Thr Val Ser Tyr Thr Asp Ile Asp Phe Ser Ser 20 25 30 Tyr Met Gln Gln Ile Phe Lys Ile Arg Gln Gly Glu Leu Asp Tyr Ser 35 40 45 Asn Ile Phe Gly Asp Thr Gly Pro Leu Val Tyr Pro Ala Gly His Val 50 55 60 His Ala Tyr Ser Val Leu Ser Trp Tyr Ser Asp Gly Gly Glu Asp Val 65 70 75 80 Ser Phe Val Gln Gln Ala Phe Gly Trp Leu Tyr Leu Gly Cys Leu Leu 85 90

95 Leu Ser Ile Ser Ser Tyr Phe Phe Ser Gly Leu Gly Lys Ile Pro Pro 100 105 110 Val Tyr Phe Val Leu Leu Val Ala Ser Lys Arg Leu His Ser Ile Phe 115 120 125 Val Leu Arg Leu Phe Asn Asp Cys Leu Thr Thr Phe Leu Met Leu Ala 130 135 140 Thr Ile Ile Ile Leu Gln Gln Ala Ser Ser Trp Arg Lys Asp Gly Thr 145 150 155 160 Thr Ile Pro Leu Ser Val Pro Asp Ala Ala Asp Thr Tyr Ser Leu Ala 165 170 175 Ile Ser Val Lys Met Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Cys Asp Glu Asn Leu Ile Lys Ala Leu Ala Pro 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Ser Phe Ile 210 215 220 Leu Pro Leu His Tyr Asp Asp Gln Ala Asn Glu Ile Arg Ser Ala Tyr 225 230 235 240 Phe Arg Gln Ala Phe Asp Phe Ser Arg Gln Phe Leu Tyr Lys Trp Thr 245 250 255 Val Asn Trp Arg Phe Leu Ser Gln Glu Thr Phe Asn Asn Val His Phe 260 265 270 His Gln Leu Leu Phe Ala Leu His Ile Ile Thr Leu Val Leu Phe Ile 275 280 285 Pro Leu Gly Arg Phe Val Leu Asp Ile Phe Lys Phe Trp Lys Pro Thr 290 295 300 Leu Ser Pro Thr Asn Ile Ile Asn Asp Pro Glu Arg Ser Pro Asp Phe 305 310 315 320 Val Tyr Thr Val Met Ala Thr Thr Asn Leu Ile Gly Val Leu Phe Ala 325 330 335 Arg Ser Leu His Tyr Gln Phe Leu Ser Trp Tyr Ala Phe Ser Leu Pro 340 345 350 Tyr Leu Leu Tyr Lys Ala Arg Leu Asn Phe Ile Ala Ser Ile Ile Val 355 360 365 Tyr Ala Ala His Glu Tyr Cys Trp Leu Val Phe Pro Ala Thr Glu Gln 370 375 380 Ser Ser Ala 385 30390PRTPichia pastorisMOD_RES(176)..(190)Variable amino acid 30Leu Trp Leu Ala Asp Ser Ile Val Ile Lys Val Ile Ile Gly Thr Val 1 5 10 15 Ser Tyr Thr Asp Ile Asp Phe Ser Ser Tyr Met Gln Gln Ile Phe Lys 20 25 30 Ile Arg Gln Gly Glu Leu Asp Tyr Ser Asn Ile Phe Gly Asp Thr Gly 35 40 45 Pro Leu Val Tyr Pro Ala Gly His Val His Ala Tyr Ser Val Leu Ser 50 55 60 Trp Tyr Ser Asp Gly Gly Glu Asp Val Ser Phe Val Gln Gln Ala Phe 65 70 75 80 Gly Trp Leu Tyr Leu Gly Cys Leu Leu Leu Ser Ile Ser Ser Tyr Phe 85 90 95 Phe Ser Gly Leu Gly Lys Ile Pro Pro Val Tyr Phe Val Leu Leu Val 100 105 110 Ala Ser Lys Arg Leu His Ser Ile Phe Val Leu Arg Leu Phe Asn Asp 115 120 125 Cys Leu Thr Thr Phe Leu Met Leu Ala Thr Ile Ile Ile Leu Gln Gln 130 135 140 Ala Ser Ser Trp Arg Lys Asp Gly Thr Thr Ile Pro Leu Ser Val Pro 145 150 155 160 Asp Ala Ala Asp Thr Tyr Ser Leu Ala Ile Ser Val Lys Met Asn Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Asp 180 185 190 Glu Asn Leu Ile Lys Ala Leu Ala Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Tyr Ser Phe Ile Leu Pro Leu His Tyr Asp Asp 210 215 220 Gln Ala Asn Glu Ile Arg Ser Ala Tyr Phe Arg Gln Ala Phe Asp Phe 225 230 235 240 Ser Arg Gln Phe Leu Tyr Lys Trp Thr Val Asn Trp Arg Phe Leu Ser 245 250 255 Gln Glu Thr Phe Asn Asn Val His Phe His Gln Leu Leu Phe Ala Leu 260 265 270 His Ile Ile Thr Leu Val Leu Phe Ile Leu Lys Phe Leu Ser Pro Lys 275 280 285 Asn Ile Gly Lys Pro Leu Gly Arg Phe Val Leu Asp Ile Phe Lys Phe 290 295 300 Trp Lys Pro Thr Leu Ser Pro Thr Asn Ile Ile Asn Asp Pro Glu Arg 305 310 315 320 Ser Pro Asp Phe Val Tyr Thr Val Met Ala Thr Thr Asn Leu Ile Gly 325 330 335 Val Leu Phe Ala Arg Ser Leu His Tyr Gln Phe Leu Ser Trp Tyr Ala 340 345 350 Phe Ser Leu Pro Tyr Leu Leu Tyr Lys Ala Arg Leu Asn Phe Ile Ala 355 360 365 Ser Ile Ile Val Tyr Ala Ala His Glu Tyr Cys Trp Leu Val Phe Pro 370 375 380 Ala Thr Glu Gln Ser Ser 385 390 31390PRTPichia pastorisMOD_RES(176)..(190)Variable amino acid 31Leu Trp Leu Ala Asp Ser Ile Val Ile Lys Val Ile Ile Gly Thr Val 1 5 10 15 Ser Tyr Thr Asp Ile Asp Phe Ser Ser Tyr Met Gln Gln Ile Phe Lys 20 25 30 Ile Arg Gln Gly Glu Leu Asp Tyr Ser Asn Ile Phe Gly Asp Thr Gly 35 40 45 Pro Leu Val Tyr Pro Ala Gly His Val His Ala Tyr Ser Val Leu Ser 50 55 60 Trp Tyr Ser Asp Gly Gly Glu Asp Val Ser Phe Val Gln Gln Ala Phe 65 70 75 80 Gly Trp Leu Tyr Leu Gly Cys Leu Leu Leu Ser Ile Ser Ser Tyr Phe 85 90 95 Phe Ser Gly Leu Gly Lys Ile Pro Pro Val Tyr Phe Val Leu Leu Val 100 105 110 Ala Ser Lys Arg Leu His Ser Ile Phe Val Leu Arg Leu Phe Asn Asp 115 120 125 Cys Leu Thr Thr Phe Leu Met Leu Ala Thr Ile Ile Ile Leu Gln Gln 130 135 140 Ala Ser Ser Trp Arg Lys Asp Gly Thr Thr Ile Pro Leu Ser Val Pro 145 150 155 160 Asp Ala Ala Asp Thr Tyr Ser Leu Ala Ile Ser Val Lys Met Asn Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Asp 180 185 190 Glu Asn Leu Ile Lys Ala Leu Ala Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Tyr Ser Phe Ile Leu Pro Leu His Tyr Asp Asp 210 215 220 Gln Ala Asn Glu Ile Arg Ser Ala Tyr Phe Arg Gln Ala Phe Asp Phe 225 230 235 240 Ser Arg Gln Phe Leu Tyr Lys Trp Thr Val Asn Trp Arg Phe Leu Ser 245 250 255 Gln Glu Thr Phe Asn Asn Val His Phe His Gln Leu Leu Phe Ala Leu 260 265 270 His Ile Ile Thr Leu Val Leu Phe Ile Leu Lys Phe Leu Ser Pro Lys 275 280 285 Asn Ile Gly Lys Pro Leu Gly Arg Phe Val Leu Asp Ile Phe Lys Phe 290 295 300 Trp Lys Pro Thr Leu Ser Pro Thr Asn Ile Ile Asn Asp Pro Glu Arg 305 310 315 320 Ser Pro Asp Phe Val Tyr Thr Val Met Ala Thr Thr Asn Leu Ile Gly 325 330 335 Val Leu Phe Ala Arg Ser Leu His Tyr Gln Phe Leu Ser Trp Tyr Ala 340 345 350 Phe Ser Leu Pro Tyr Leu Leu Tyr Lys Ala Arg Leu Asn Phe Ile Ala 355 360 365 Ser Ile Ile Val Tyr Ala Ala His Glu Tyr Cys Trp Leu Val Phe Pro 370 375 380 Ala Thr Glu Gln Ser Ser 385 390 32428DNAKluyveromyces lactis 32tttgtttaca agctgatacc aacgaacatg aatacaccgg caggtttact gaagattggc 60aaagctaacc ttttacatcc ttttaccgat gctgtattca gtgcgatgag agtaaacgca 120gaacaaattg catacatttt acttgttacc aattacattg gagtactatt tgctcgatca 180ttacactacc aattcctatc ttggtaccat tggacgttac cagtactatt gaattgggcc 240aatgttccgt atccgctatg tgtgctatgg tacctaacac atgagtggtg ctggaacagc 300tatccgccaa acgctactgc atccacactg ctacacgcgt gtaacacata ctgttattgg 360ctgtattctt aagaggaccc gcaaactcga aaagtggtga taacgaaaca acacacgaga 420aagctgag 428331668DNASaccharomyces cerevisiae 33atgaattgca aggcggtaac cattagttta ttactgttgt tatttttaac aagagtatat 60attcagccga cattctcgtt aatttcagat tgcgatgaaa cttttaatta ttgggaacca 120ttaaatttat tggtacgtgg atttggtaaa caaacctggg aatattcacc cgagtattct 180attagatcat gggctttctt attacctttt tactgtattc tttatccagt aaacaaattt 240actgacctag aaagtcattg gaactttttc atcacaagag catgcttagg cttttttagt 300tttatcatgg aatttaaact acatcgtgaa attgcaggca gcttggcatt gcaaatcgca 360aatatttgga ttattttcca attgtttaat ccgggctggt tccatgcatc tgtggaatta 420ttgccttctg ccgttgccat gttgttgtat gtaggtgcca ccagacactc tctacgctat 480ctgtccactg ggtctacttc taactttacg aaaagtttag cgtacaattt cctggctagt 540atactaggct ggccatttgt tttaatttta agcttgccat tatgtttaca ttaccttttc 600aaccatagaa ttatttctac catcagaacc gcattcgact gctgtttgat attttcattg 660actgcatttg ctgtgattgt cactgacagt atattttacg ggaagcttgc tcctgtatca 720tggaacatct tattttacaa tgtcattaat gcaagtgagg aatctggccc aaatattttc 780ggggttgagc catggtacta ctatccacta aatttgttac tgaatttccc actgcctgtg 840ctagttttag ctattttggg aattttccat ttgagattat ggccattatg ggcatcatta 900ttcacatgga ttgccgtttt cactcaacaa cctcacaaag aggaaagatt tctctatcca 960atttacgggt taataacttt gagtgcaagt atcgcctttt acaaagtgtt gaatctattc 1020aatagaaagc cgattcttaa aaaaggtata aagttgtcag ttttattaat tgttgcaggc 1080caggcaatgt cacggatagt ggctttggtg aacaattaca cagctcctat agccgtctac 1140gagcaatttt cttcactaaa tcaaggtggt gtgaaggcac cggtagtgaa tgtatgtacg 1200ggacgtgaat ggtatcactt cccaagttct ttcctgctgc cagataatca taggctaaaa 1260tttgttaaat ctggatttga tggtcttctt ccaggtgatt ttccagagag tggttctatt 1320ttcaaaaaga ttagaacttt acctaaggga atgaataaca agaatatata tgataccggt 1380aaagagtggc cgatcactag atgtgattat tttattgaca tcgtcgcccc aataaattta 1440acaaaagacg ttttcaaccc tctacatctg atggataact ggaataagct ggcatgtgct 1500gcattcatcg acggtgaaaa ttctaagatt ttgggtagag cattttacgt accggagcca 1560atcaaccgaa tcatgcaaat agttttacca aaacaatgga atcaagtgta cggtgttcgt 1620tacattgatt actgtttgtt tgaaaaacca actgagacta ctaattga 166834600DNAPichia pastoris 34tggccttcct gtctgctcga tacttccttt tacagtaacc aacatacatg ttctccaaca 60tgctcttgta tgtattggcc tattctatct tgagacttga tatcaacctt ctatggtatt 120atttcagact gtgatgaagt gttcaactac tgggagccac tcaacttcat gcttagaggg 180tttggaaaac agacttggga gtattctcca gagtatgcca tccgatcttg gtcctatcta 240gtgccacttt ggatagcagg ctatccacca ttgttcctgg atatcccttc ttactacttt 300ttctactttt tcagactact gctggttatt ttttcattgg ttgcagaagt caagttgtac 360catagtttga agaaaaatgt cagcagtaag atcagtttct ggtaccttct atttacaacc 420gttgctccag gaatgtctca tagcacgata gccttattac catcctcttt tgctatggtt 480tgtcacactt ttgccattag atacgtcatt gattacctac aattaccaac attaatgcgc 540acaatcagag agactgctgc catctcacca gctcacaaac aacaactagc caactctctc 60035140PRTPichia pastorisMOD_RES(65)..(71)Variable amino acid 35Ile Ser Thr Phe Tyr Gly Ile Ile Ser Asp Cys Asp Glu Val Phe Asn 1 5 10 15 Tyr Trp Glu Pro Leu Asn Phe Met Leu Arg Gly Phe Gly Lys Gln Thr 20 25 30 Trp Glu Tyr Ser Pro Glu Tyr Ala Ile Arg Ser Trp Ser Tyr Leu Val 35 40 45 Pro Leu Trp Ile Ala Gly Tyr Pro Pro Leu Phe Leu Asp Ile Pro Ser 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Leu Leu Val Ile Phe Ser Leu 65 70 75 80 Val Ala Glu Val Lys Leu Tyr His Ser Leu Lys Lys Asn Val Ser Ser 85 90 95 Lys Ile Ser Phe Trp Tyr Leu Leu Phe Thr Thr Val Ala Pro Gly Met 100 105 110 Ser His Ser Thr Ile Ala Leu Leu Pro Ser Ser Phe Ala Met Val Cys 115 120 125 His Thr Phe Ala Ile Arg Tyr Val Ile Asp Tyr Leu 130 135 140 36127PRTPichia pastorisMOD_RES(66)..(72)Variable amino acid 36Leu Ile Ser Thr Phe Tyr Gly Ile Ile Ser Asp Cys Asp Glu Val Phe 1 5 10 15 Asn Tyr Trp Glu Pro Leu Asn Phe Met Leu Arg Gly Phe Gly Lys Gln 20 25 30 Thr Trp Glu Tyr Ser Pro Glu Tyr Ala Ile Arg Ser Trp Ser Tyr Leu 35 40 45 Val Pro Leu Trp Ile Ala Gly Tyr Pro Pro Leu Phe Leu Asp Ile Pro 50 55 60 Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Leu Leu Val Ile Phe Ser 65 70 75 80 Leu Val Ala Glu Val Lys Leu Tyr His Ser Leu Lys Lys Asn Val Ser 85 90 95 Ser Lys Ile Ser Phe Trp Tyr Leu Leu Phe Thr Thr Val Ala Pro Gly 100 105 110 Met Ser His Ser Thr Ile Ala Leu Leu Pro Ser Ser Phe Ala Met 115 120 125 37157PRTPichia pastorisMOD_RES(66)..(72)Variable amino acid 37Leu Ile Ser Thr Phe Tyr Gly Ile Ile Ser Asp Cys Asp Glu Val Phe 1 5 10 15 Asn Tyr Trp Glu Pro Leu Asn Phe Met Leu Arg Gly Phe Gly Lys Gln 20 25 30 Thr Trp Glu Tyr Ser Pro Glu Tyr Ala Ile Arg Ser Trp Ser Tyr Leu 35 40 45 Val Pro Leu Trp Ile Ala Gly Tyr Pro Pro Leu Phe Leu Asp Ile Pro 50 55 60 Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Leu Leu Val Ile Phe Ser 65 70 75 80 Leu Val Ala Glu Val Lys Leu Tyr His Ser Leu Lys Lys Asn Val Ser 85 90 95 Ser Lys Ile Ser Phe Trp Tyr Leu Leu Phe Thr Thr Val Ala Pro Gly 100 105 110 Met Ser His Ser Thr Ile Ala Leu Leu Pro Ser Ser Phe Ala Met Val 115 120 125 Cys His Thr Phe Ala Ile Arg Tyr Val Ile Asp Tyr Leu Gln Leu Pro 130 135 140 Thr Leu Met Arg Thr Ile Arg Glu Thr Ala Ala Ile Ser 145 150 155 38141PRTPichia pastorisMOD_RES(80)..(86)Variable amino acid 38Ser Pro Thr Cys Ser Cys Met Tyr Trp Pro Ile Leu Ser Asp Leu Ile 1 5 10 15 Ser Thr Phe Tyr Gly Ile Ile Ser Asp Cys Asp Glu Val Phe Asn Tyr 20 25 30 Trp Glu Pro Leu Asn Phe Met Leu Arg Gly Phe Gly Lys Gln Thr Trp 35 40 45 Glu Tyr Ser Pro Glu Tyr Ala Ile Arg Ser Trp Ser Tyr Leu Val Pro 50 55 60 Leu Trp Ile Ala Gly Tyr Pro Pro Leu Phe Leu Asp Ile Pro Ser Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Leu Leu Val Ile Phe Ser Leu Val 85 90 95 Ala Glu Val Lys Leu Tyr His Ser Leu Lys Lys Asn Val Ser Ser Lys 100 105 110 Ile Ser Phe Trp Tyr Leu Leu Phe Thr Thr Val Ala Pro Gly Met Ser 115 120 125 His Ser Thr Ile Ala Leu Leu Pro Ser Ser Phe Ala Met 130 135 140 39141PRTPichia pastorisMOD_RES(80)..(86)Variable amino acid 39Ser Pro Thr Cys Ser Cys Met Tyr Trp Pro Ile Leu Ser Asp Leu Ile 1 5 10 15 Ser Thr Phe Tyr Gly Ile Ile Ser Asp Cys Asp Glu Val Phe Asn Tyr 20 25 30 Trp Glu Pro Leu Asn Phe Met Leu Arg Gly Phe Gly Lys Gln Thr Trp 35 40 45 Glu Tyr Ser Pro Glu Tyr Ala Ile Arg Ser Trp Ser Tyr Leu Val Pro 50 55 60 Leu Trp Ile Ala Gly Tyr Pro Pro Leu Phe Leu Asp Ile Pro Ser Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Arg Leu Leu Leu Val Ile Phe Ser Leu Val 85 90 95 Ala Glu Val Lys Leu Tyr His Ser Leu Lys Lys Asn Val Ser Ser Lys 100 105 110 Ile Ser Phe Trp Tyr Leu Leu Phe Thr Thr Val Ala Pro Gly Met Ser 115 120 125 His Ser Thr Ile Ala Leu Leu Pro Ser Ser Phe Ala Met 130 135 140 401656DNASaccharomyces cerevisiae 40atgcgttggt ctgtccttga tacagtgcta ttgaccgtga tttcctttca tctaatccaa 60gctccattca ccaaggtgga agagagtttt aatattcaag ccattcatga tattttaacc 120tacagcgtat ttgatatctc ccaatatgac cacttgaaat ttcctggagt agtccctaga

180acattcgttg gtgctgtgat tattgcaatg ctttcgagac cttatcttta cttgagttct 240ttgatccaaa cttccaggcc tacgtctata gatgttcaat tggtcgttag ggggattgtt 300ggcctcacca atgggctttc ttttatctat ttaaagaatt gtttgcaaga tatgtttgat 360gaaatcactg aaaagaaaaa ggaagaaaat gaagacaagg atatatacat ttacgatagc 420gctggtacat ggtttctttt atttttaatt ggcagtttcc acctcatgtt ctacagcact 480aggactctgc ctaattttgt catgactctg cctctaacca acgtcgcatt ggggtgggtt 540ttattgggtc gttataatgc agctatattc ctatctgcgc tcgtggcaat tgtatttaga 600ctggaagtgt cagctctcag tgctggtatt gctctattta gcgtcatctt caagaagatt 660tctttattcg atgctatcaa attcggtatc tttggcttgg gacttggttc cgccatcagt 720atcaccgttg attcatattt ctggcaagaa tggtgtctac ctgaggtaga tggtttcttg 780ttcaacgtgg ttgcgggtta cgcttccaag tggggtgtgg agccagttac tgcttatttc 840acgcattact tgagaatgat gtttatgcca ccaactgttt tactattgaa ttacttcggc 900tataaattag cacctgcaaa attaaaaatt gtctcactag catctctttt ccacattatc 960gtcttatcct ttcaacctca caaagaatgg agattcatca tctacgctgt tccatctatc 1020atgttgctag gtgccacagg agcagcacat ctatgggaga atatgaaagt aaaaaagatt 1080accaatgttt tatgtttggc tatattgccc ttatctataa tgacctcctt tttcatttca 1140atggcgttct tgtatatatc aagaatgaat tatccaggcg gcgaggcttt aacttctttt 1200aatgacatga ttgtggaaaa aaatattaca aacgctacag ttcatatcag catacctcct 1260tgcatgacag gtgtcacttt atttggtgaa ttgaactacg gtgtgtacgg catcaattac 1320gataagactg aaaatacgac tttactgcag gaaatgtggc cctcctttga tttcttgatc 1380acccacgagc caaccgcctc tcaattgcca ttcgagaata agactaccaa ccattgggag 1440ctagttaaca caacaaagat gtttactgga tttgacccaa cctacattaa gaactttgtt 1500ttccaagaga gagtgaatgt tttgtctcta ctcaaacaga tcattttcga caagacccct 1560accgtttttt tgaaagaatt gacggccaat tcgattgtta aaagcgatgt cttcttcacc 1620tataagagaa tcaaacaaga tgaaaaaact gattga 165641840DNAPichia pastoris 41tcggtcgaga atgataactg aagaactcaa aatctctcac actttcatcg ttactgtact 60ggcaatcatt gcatttcagc ctcataaaga atggagattt atagtttaca ttgttccacc 120acttgtcatc accatatcta cagtacttgc acaactaccc aggagattca caatcgtcaa 180agttgctgtt tttctcctaa gtttcggctc tttgctcata tccctgtcgt ttcttttcat 240ctcatcgtat aactaccctg ggggtgaagc tttacagcat ttgaacgaga aactccttct 300actggaccaa agttccctac ctgttgatat taaggttcat atggatgtcc ctgcatgcat 360gactggggtg actttatttg gttacttgga taactcaaaa ttgaacaatt taagaattgt 420ctatgataaa acagaagacg agtcgctgga cacaatctgg gattctttca attatgtcat 480ctccgaaatt gacttggatt cttcgactgc tcccaaatgg gagggggatt ggctgaagat 540tgatgttgtc caaggctaca acggcatcaa taaacaatct atcaaaaata caattttcaa 600ttatggaata cttaaacgga tgataagaga cgcaaccaaa cttgatgttg gatttattcg 660tacggtcttt cgatccttca taaaatttga tgataaatta ttcatttatg agaggagcag 720tcaaacctga aaatatatac ctcatttgtt caatttggtg taaagagtgt ggcggataga 780cttcttgtaa atcaggaaag ctacaattcc aattgctgca aaaaatacca atgcccataa 84042239PRTPichia pastorisMOD_RES(62)..(80)Variable amino acid 42Arg Met Ile Thr Glu Glu Leu Lys Ile Ser His Thr Phe Ile Val Thr 1 5 10 15 Val Leu Ala Ile Ile Ala Phe Gln Pro His Lys Glu Trp Arg Phe Ile 20 25 30 Val Tyr Ile Val Pro Pro Leu Val Ile Thr Ile Ser Thr Val Leu Ala 35 40 45 Gln Leu Pro Arg Arg Phe Thr Ile Val Lys Val Ala Val Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Tyr Asn Tyr Pro Gly Gly Glu Ala Leu Gln His Leu Asn Glu Lys Leu 85 90 95 Leu Leu Leu Asp Gln Ser Ser Leu Pro Val Asp Ile Lys Val His Met 100 105 110 Asp Val Pro Ala Cys Met Thr Gly Val Thr Leu Phe Gly Tyr Leu Asp 115 120 125 Asn Ser Lys Leu Asn Asn Leu Arg Ile Val Tyr Asp Lys Thr Glu Asp 130 135 140 Glu Ser Leu Asp Thr Ile Trp Asp Ser Phe Asn Tyr Val Ile Ser Glu 145 150 155 160 Ile Asp Leu Asp Ser Ser Thr Ala Pro Lys Trp Glu Gly Asp Trp Leu 165 170 175 Lys Ile Asp Val Val Gln Gly Tyr Asn Gly Ile Asn Lys Gln Ser Ile 180 185 190 Lys Asn Thr Ile Phe Asn Tyr Gly Ile Leu Lys Arg Met Ile Arg Asp 195 200 205 Ala Thr Lys Leu Asp Val Gly Phe Ile Arg Thr Val Phe Arg Ser Phe 210 215 220 Ile Lys Phe Asp Asp Lys Leu Phe Ile Tyr Glu Arg Ser Ser Gln 225 230 235 43141PRTPichia pastorisMOD_RES(43)..(61)Variable amino acid 43Ile Ile Ala Phe Gln Pro His Lys Glu Trp Arg Phe Ile Val Tyr Ile 1 5 10 15 Val Pro Pro Leu Val Ile Thr Ile Ser Thr Val Leu Ala Gln Leu Pro 20 25 30 Arg Arg Phe Thr Ile Val Lys Val Ala Val Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Asn Tyr 50 55 60 Pro Gly Gly Glu Ala Leu Gln His Leu Asn Glu Lys Leu Leu Leu Leu 65 70 75 80 Asp Gln Ser Ser Leu Pro Val Asp Ile Lys Val His Met Asp Val Pro 85 90 95 Ala Cys Met Thr Gly Val Thr Leu Phe Gly Tyr Leu Asp Asn Ser Lys 100 105 110 Leu Asn Asn Leu Arg Ile Val Tyr Asp Lys Thr Glu Asp Glu Ser Leu 115 120 125 Asp Thr Ile Trp Asp Ser Phe Asn Tyr Val Ile Ser Glu 130 135 140 44143PRTPichia pastorisMOD_RES(45)..(63)Variable amino acid 44Leu Ala Ile Ile Ala Phe Gln Pro His Lys Glu Trp Arg Phe Ile Val 1 5 10 15 Tyr Ile Val Pro Pro Leu Val Ile Thr Ile Ser Thr Val Leu Ala Gln 20 25 30 Leu Pro Arg Arg Phe Thr Ile Val Lys Val Ala Val Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 50 55 60 Asn Tyr Pro Gly Gly Glu Ala Leu Gln His Leu Asn Glu Lys Leu Leu 65 70 75 80 Leu Leu Asp Gln Ser Ser Leu Pro Val Asp Ile Lys Val His Met Asp 85 90 95 Val Pro Ala Cys Met Thr Gly Val Thr Leu Phe Gly Tyr Leu Asp Asn 100 105 110 Ser Lys Leu Asn Asn Leu Arg Ile Val Tyr Asp Lys Thr Glu Asp Glu 115 120 125 Ser Leu Asp Thr Ile Trp Asp Ser Phe Asn Tyr Val Ile Ser Glu 130 135 140 451635DNASaccharomyces cerevisiae 45atggccattg gcaaaaggtt actggtgaac aaaccagcag aagaatcatt ttatgcttct 60ccaatgtatg attttttgta tccgtttagg ccagtgggga accaatggct gccagaatat 120attatctttg tatgtgctgt aatactgagg tgcacaattg gacttggtcc atattctggg 180aaaggcagtc caccgctgta cggcgatttt gaggctcaga gacattggat ggaaattacg 240caacatttac cgctttctaa gtggtactgg tatgatttgc aatactgggg attggactat 300ccaccattaa cagcatttca ttcgtacctt ctgggcctaa ttggatcttt tttcaatcca 360tcttggtttg cactagaaaa gtcacgtggc tttgaatccc ccgataatgg cctgaaaaca 420tatatgcgtt ctactgtcat cattagcgac atattgtttt actttcctgc agtaatatac 480tttactaagt ggcttggtag atatcgaaac cagtcgccca taggacaatc tattgcggca 540tcagcgattt tgttccaacc ttcattaatg ctcattgacc atgggcactt tcaatataat 600tcagtcatgc ttggccttac tgcttatgcc ataaataact tattagatga gtattatgct 660atggcggccg tttgttttgt cctatccatt tgttttaaac aaatggcatt gtattatgca 720ccgatttttt ttgcttatct attaagtcga tcattgctgt tccccaaatt taacatagct 780agattgacgg ttattgcgtt tgcaacactc gcaacttttg ctataatatt tgcgccatta 840tatttcttgg gaggaggatt aaagaatatt caccaatgta ttcacaggat attccctttt 900gccaggggca tcttcgaaga caaggttgct aacttctggt gcgttacgaa cgtgtttgta 960aaatacaagg aaagattcac tatacaacaa ctccagctat attcattgat tgccaccgtg 1020attggtttct taccagccat gataatgaca ttacttcatc ccaaaaagca tcttctccca 1080tacgtgttaa tcgcatgttc gatgtccttt tttcttttta gctttcaagt acatgagaaa 1140actatcctca tcccactttt gcctattaca ctactctact cctctactga ttggaatgtt 1200ctatctcttg taagttggat aaacaatgtg gctttgttta cgctatggcc tttgttgaaa 1260aaggacggtc ttcatttaca gtatgccgta tctttcttac taagcaattg gctgattgga 1320aatttcagtt ttattacacc aaggttcttg ccaaaatctt taactcctgg cccttctatc 1380agcagcatca atagcgacta tagaagaaga agcttactgc catataatgt ggtttggaaa 1440agttttatca taggaacgta tattgctatg ggcttttatc atttcttaga tcaatttgta 1500gcacctccat cgaaatatcc agacttgtgg gtgttgttga actgtgctgt tgggttcatt 1560tgctttagca tattttggct atggtcttat tacaagatat tcacttccgg tagcaaatcc 1620atgaaggact tgtag 1635461644DNAPichia pastoris 46atgccacata aaagaacgcc ctctagcagt ctgctgtatg caagaattcc agggatctct 60tttgaaaact ctccggtgtt tgattttttg tctccttttg gacccgctcc taatcaatgg 120gtagcacgat acatcatcat catctttgca attctcatca gattggcagt tgggctgggc 180tcctattccg gcttcaacac ccctccaatg tatggggatt ttgaagctca gaggcattgg 240atggaaatta ctcagcattt atccatagaa aaatggtact tctacgactt gcaatattgg 300gggcttgact atcctccctt gacagccttt cattcatact tctttggcaa attaggcagc 360ttcatcaatc cagcatggtt tgctttagac gtctccagag ggtttgaatc agtggatcta 420aaatcgtaca tgagggcgac cgcaattctc agtgagctgt tatgttttat tccagctgtc 480atttggtatt gtcgttggat gggacttaac tacttcaatc aaaacgccat tgagcaaact 540ataatagcgt ctgctattct tttcaatcca tctttaatta tcatagatca tggccacttc 600cagtacaact cagttatgct aggttttgct ttattatcca tattaaatct gttgtacgat 660aattttgcat tagcggctat ttttttcgtt ctttcaataa gctttaagca aatggctctc 720tattatagcc ccatcatgtt tttttacatg ctgagtgtga gttgttggcc tttgaaaaac 780ttcaacttgt tgagattggc tactatcagt attgcagtac tcttgacttt tgcaactcta 840ttactgcctt ttgtattagt agatgggatg tcacaaattg gccaaatatt attcagagtt 900ttcccgtttt caagaggctt gtttgaggat aaggtggcca acttttggtg tacaacgaat 960atactggtaa agtacaaaca gttattcact gacaaaaccc ttactaggat atcgctagta 1020gcaactttga ttgcaattag tccgtcttgc ttcatcattt ttactcaccc aaagaaggtt 1080ttactaccgt gggcttttgc tgcttgctct tgggcgttct atcttttctc tttccaagtc 1140cacgagaaat cagttttagt tccattgatg cctaccactc tattactggt agaaaaagac 1200ttggacatca tctcaatggt ctgctggatt tctaatattg ccttcttcag catgtggcct 1260ctattaaaaa gagacgggct ggctttggaa tattttgtct tgggaatatt gagtaattgg 1320ctgattggaa acctcaattg gattagtaaa tggcttgtcc ccagtttcct gattccaggg 1380cctactctct ccaaaaaagt tcctaaaaga gatactaaaa cagttgttca tactcactgg 1440ttttgggggt cagtaacatt cgtttcatac ctcggagcta cagttatcca gttcgtagat 1500tggctgtacc ttccacctgc caagtatcca gatttgtggg ttattttgaa cactacattg 1560tcgtttgctt gtttcgggtt gttttggcta tggattaact acaatctgta cattttgcgt 1620gattttaagc ttaaagatgc ttag 164447527PRTPichia pastorisMOD_RES(23)..(37)Variable amino acid 47Ser Phe Glu Asn Ser Pro Val Phe Asp Phe Leu Ser Pro Phe Gly Pro 1 5 10 15 Ala Pro Asn Gln Trp Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Val Gly Leu Gly Ser Tyr Ser Gly Phe Asn Thr 35 40 45 Pro Pro Met Tyr Gly Asp Phe Glu Ala Gln Arg His Trp Met Glu Ile 50 55 60 Thr Gln His Leu Ser Ile Glu Lys Trp Tyr Phe Tyr Asp Leu Gln Tyr 65 70 75 80 Trp Gly Leu Asp Tyr Pro Pro Leu Thr Ala Phe His Ser Tyr Phe Phe 85 90 95 Gly Lys Leu Gly Ser Phe Ile Asn Pro Ala Trp Phe Ala Leu Asp Val 100 105 110 Ser Arg Gly Phe Glu Ser Val Asp Leu Lys Ser Tyr Met Arg Ala Thr 115 120 125 Ala Ile Leu Ser Glu Leu Leu Cys Phe Ile Pro Ala Val Ile Trp Tyr 130 135 140 Cys Arg Trp Met Gly Leu Asn Tyr Phe Asn Gln Asn Ala Ile Glu Gln 145 150 155 160 Thr Ile Ile Ala Ser Ala Ile Leu Phe Asn Pro Ser Leu Ile Ile Ile 165 170 175 Asp His Gly His Phe Gln Tyr Asn Ser Val Met Leu Gly Phe Ala Leu 180 185 190 Leu Ser Ile Leu Asn Leu Leu Tyr Asp Asn Phe Ala Leu Ala Ala Ile 195 200 205 Phe Phe Val Leu Ser Ile Ser Phe Lys Gln Met Ala Leu Tyr Tyr Ser 210 215 220 Pro Ile Met Phe Phe Tyr Met Leu Ser Val Ser Cys Trp Pro Leu Lys 225 230 235 240 Asn Phe Asn Leu Leu Arg Leu Ala Thr Ile Ser Ile Ala Val Leu Leu 245 250 255 Thr Phe Ala Thr Leu Leu Leu Pro Phe Val Leu Val Asp Gly Met Ser 260 265 270 Gln Ile Gly Gln Ile Leu Phe Arg Val Phe Pro Phe Ser Arg Gly Leu 275 280 285 Phe Glu Asp Lys Val Ala Asn Phe Trp Cys Thr Thr Asn Ile Leu Val 290 295 300 Lys Tyr Lys Gln Leu Phe Thr Asp Lys Thr Leu Thr Arg Ile Ser Leu 305 310 315 320 Val Ala Thr Leu Ile Ala Ile Ser Pro Ser Cys Phe Ile Ile Phe Thr 325 330 335 His Pro Lys Lys Val Leu Leu Pro Trp Ala Phe Ala Ala Cys Ser Trp 340 345 350 Ala Phe Tyr Leu Phe Ser Phe Gln Val His Glu Lys Ser Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Lys Asp Leu Asp Ile 370 375 380 Ile Ser Met Val Cys Trp Ile Ser Asn Ile Ala Phe Phe Ser Met Trp 385 390 395 400 Pro Leu Leu Lys Arg Asp Gly Leu Ala Leu Glu Tyr Phe Val Leu Gly 405 410 415 Ile Leu Ser Asn Trp Leu Ile Gly Asn Leu Asn Trp Ile Ser Lys Trp 420 425 430 Leu Val Pro Ser Phe Leu Ile Pro Gly Pro Thr Leu Ser Lys Lys Val 435 440 445 Pro Lys Arg Asp Thr Lys Thr Val Val His Thr His Trp Phe Trp Gly 450 455 460 Ser Val Thr Phe Val Ser Tyr Leu Gly Ala Thr Val Ile Gln Phe Val 465 470 475 480 Asp Trp Leu Tyr Leu Pro Pro Ala Lys Tyr Pro Asp Leu Trp Val Ile 485 490 495 Leu Asn Thr Thr Leu Ser Phe Ala Cys Phe Gly Leu Phe Trp Leu Trp 500 505 510 Ile Asn Tyr Asn Leu Tyr Ile Leu Arg Asp Phe Lys Leu Lys Asp 515 520 525 48511PRTPichia pastorisMOD_RES(22)..(36)Variable amino acid 48Phe Glu Asn Ser Pro Val Phe Asp Phe Leu Ser Pro Phe Gly Pro Ala 1 5 10 15 Pro Asn Gln Trp Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Val Gly Leu Gly Ser Tyr Ser Gly Phe Asn Thr Pro 35 40 45 Pro Met Tyr Gly Asp Phe Glu Ala Gln Arg His Trp Met Glu Ile Thr 50 55 60 Gln His Leu Ser Ile Glu Lys Trp Tyr Phe Tyr Asp Leu Gln Tyr Trp 65 70 75 80 Gly Leu Asp Tyr Pro Pro Leu Thr Ala Phe His Ser Tyr Phe Phe Gly 85 90 95 Lys Leu Gly Ser Phe Ile Asn Pro Ala Trp Phe Ala Leu Asp Val Ser 100 105 110 Arg Gly Phe Glu Ser Val Asp Leu Lys Ser Tyr Met Arg Ala Thr Ala 115 120 125 Ile Leu Ser Glu Leu Leu Cys Phe Ile Pro Ala Val Ile Trp Tyr Cys 130 135 140 Arg Trp Met Gly Leu Asn Tyr Phe Asn Gln Asn Ala Ile Glu Gln Thr 145 150 155 160 Ile Ile Ala Ser Ala Ile Leu Phe Asn Pro Ser Leu Ile Ile Ile Asp 165 170 175 His Gly His Phe Gln Tyr Asn Ser Val Met Leu Gly Phe Ala Leu Leu 180 185 190 Ser Ile Leu Asn Leu Leu Tyr Asp Asn Phe Ala Leu Ala Ala Ile Phe 195 200 205 Phe Val Leu Ser Ile Ser Phe Lys Gln Met Ala Leu Tyr Tyr Ser Pro 210 215 220 Ile Met Phe Phe Tyr Met Leu Ser Val Ser Cys Trp Pro Leu Lys Asn 225 230 235 240 Phe Asn Leu Leu Arg Leu Ala Thr Ile Ser Ile Ala Val Leu Leu Thr 245 250 255 Phe Ala Thr Leu Leu Leu Pro Phe Val Leu Val Asp Gly Met Ser Gln 260 265 270 Ile Gly Gln Ile Leu Phe Arg Val Phe Pro Phe Ser Arg Gly Leu Phe 275 280 285 Glu Asp Lys Val Ala Asn Phe Trp Cys Thr Thr Asn Ile Leu Val Lys 290 295 300 Tyr Lys Gln Leu Phe Thr Asp Lys Thr Leu Thr Arg Ile Ser Leu Val 305 310 315 320 Ala Thr Leu Ile Ala

Ile Ser Pro Ser Cys Phe Ile Ile Phe Thr His 325 330 335 Pro Lys Lys Val Leu Leu Pro Trp Ala Phe Ala Ala Cys Ser Trp Ala 340 345 350 Phe Tyr Leu Phe Ser Phe Gln Val His Glu Lys Ser Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Lys Asp Leu Asp Ile Ile 370 375 380 Ser Met Val Cys Trp Ile Ser Asn Ile Ala Phe Phe Ser Met Trp Pro 385 390 395 400 Leu Leu Lys Arg Asp Gly Leu Ala Leu Glu Tyr Phe Val Leu Gly Ile 405 410 415 Leu Ser Asn Trp Leu Ile Gly Asn Leu Asn Trp Ile Ser Lys Trp Leu 420 425 430 Val Pro Ser Phe Leu Ile Pro Gly Pro Thr Leu Ser Lys Lys Val Pro 435 440 445 Lys Arg Asp Thr Lys Thr Val Val His Thr His Trp Phe Trp Gly Ser 450 455 460 Val Thr Phe Val Ser Tyr Leu Gly Ala Thr Val Ile Gln Phe Val Asp 465 470 475 480 Trp Leu Tyr Leu Pro Pro Ala Lys Tyr Pro Asp Leu Trp Val Ile Leu 485 490 495 Asn Thr Thr Leu Ser Phe Ala Cys Phe Gly Leu Phe Trp Leu Trp 500 505 510 49477PRTPichia pastorisMOD_RES(329)..(341)Variable amino acid 49Val Gly Leu Gly Ser Tyr Ser Gly Phe Asn Thr Pro Pro Met Tyr Gly 1 5 10 15 Asp Phe Glu Ala Gln Arg His Trp Met Glu Ile Thr Gln His Leu Ser 20 25 30 Ile Glu Lys Trp Tyr Phe Tyr Asp Leu Gln Tyr Trp Gly Leu Asp Tyr 35 40 45 Pro Pro Leu Thr Ala Phe His Ser Tyr Phe Phe Gly Lys Leu Gly Ser 50 55 60 Phe Ile Asn Pro Ala Trp Phe Ala Leu Asp Val Ser Arg Gly Phe Glu 65 70 75 80 Ser Val Asp Leu Lys Ser Tyr Met Arg Ala Thr Ala Ile Leu Ser Glu 85 90 95 Leu Leu Cys Phe Ile Pro Ala Val Ile Trp Tyr Cys Arg Trp Met Gly 100 105 110 Leu Asn Tyr Phe Asn Gln Asn Ala Ile Glu Gln Thr Ile Ile Ala Ser 115 120 125 Ala Ile Leu Phe Asn Pro Ser Leu Ile Ile Ile Asp His Gly His Phe 130 135 140 Gln Tyr Asn Ser Val Met Leu Gly Phe Ala Leu Leu Ser Ile Leu Asn 145 150 155 160 Leu Leu Tyr Asp Asn Phe Ala Leu Ala Ala Ile Phe Phe Val Leu Ser 165 170 175 Ile Ser Phe Lys Gln Met Ala Leu Tyr Tyr Ser Pro Ile Met Phe Phe 180 185 190 Tyr Met Leu Ser Val Ser Cys Trp Pro Leu Lys Asn Phe Asn Leu Leu 195 200 205 Arg Leu Ala Thr Ile Ser Ile Ala Val Leu Leu Thr Phe Ala Thr Leu 210 215 220 Leu Leu Pro Phe Val Leu Val Asp Gly Met Ser Gln Ile Gly Gln Ile 225 230 235 240 Leu Phe Arg Val Phe Pro Phe Ser Arg Gly Leu Phe Glu Asp Lys Val 245 250 255 Ala Asn Phe Trp Cys Thr Thr Asn Ile Leu Val Lys Tyr Lys Gln Leu 260 265 270 Phe Thr Asp Lys Thr Leu Thr Arg Ile Ser Leu Val Ala Thr Leu Ile 275 280 285 Ala Ile Ser Pro Ser Cys Phe Ile Ile Phe Thr His Pro Lys Lys Val 290 295 300 Leu Leu Pro Trp Ala Phe Ala Ala Cys Ser Trp Ala Phe Tyr Leu Phe 305 310 315 320 Ser Phe Gln Val His Glu Lys Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Glu Lys Asp Leu Asp Ile Ile Ser Met Val Cys 340 345 350 Trp Ile Ser Asn Ile Ala Phe Phe Ser Met Trp Pro Leu Leu Lys Arg 355 360 365 Asp Gly Leu Ala Leu Glu Tyr Phe Val Leu Gly Ile Leu Ser Asn Trp 370 375 380 Leu Ile Gly Asn Leu Asn Trp Ile Ser Lys Trp Leu Val Pro Ser Phe 385 390 395 400 Leu Ile Pro Gly Pro Thr Leu Ser Lys Lys Val Pro Lys Arg Asp Thr 405 410 415 Lys Thr Val Val His Thr His Trp Phe Trp Gly Ser Val Thr Phe Val 420 425 430 Ser Tyr Leu Gly Ala Thr Val Ile Gln Phe Val Asp Trp Leu Tyr Leu 435 440 445 Pro Pro Ala Lys Tyr Pro Asp Leu Trp Val Ile Leu Asn Thr Thr Leu 450 455 460 Ser Phe Ala Cys Phe Gly Leu Phe Trp Leu Trp Ile Asn 465 470 475 50478PRTPichia pastorisMOD_RES(324)..(336)Variable amino acid 50Tyr Ser Gly Phe Asn Thr Pro Pro Met Tyr Gly Asp Phe Glu Ala Gln 1 5 10 15 Arg His Trp Met Glu Ile Thr Gln His Leu Ser Ile Glu Lys Trp Tyr 20 25 30 Phe Tyr Asp Leu Gln Tyr Trp Gly Leu Asp Tyr Pro Pro Leu Thr Ala 35 40 45 Phe His Ser Tyr Phe Phe Gly Lys Leu Gly Ser Phe Ile Asn Pro Ala 50 55 60 Trp Phe Ala Leu Asp Val Ser Arg Gly Phe Glu Ser Val Asp Leu Lys 65 70 75 80 Ser Tyr Met Arg Ala Thr Ala Ile Leu Ser Glu Leu Leu Cys Phe Ile 85 90 95 Pro Ala Val Ile Trp Tyr Cys Arg Trp Met Gly Leu Asn Tyr Phe Asn 100 105 110 Gln Asn Ala Ile Glu Gln Thr Ile Ile Ala Ser Ala Ile Leu Phe Asn 115 120 125 Pro Ser Leu Ile Ile Ile Asp His Gly His Phe Gln Tyr Asn Ser Val 130 135 140 Met Leu Gly Phe Ala Leu Leu Ser Ile Leu Asn Leu Leu Tyr Asp Asn 145 150 155 160 Phe Ala Leu Ala Ala Ile Phe Phe Val Leu Ser Ile Ser Phe Lys Gln 165 170 175 Met Ala Leu Tyr Tyr Ser Pro Ile Met Phe Phe Tyr Met Leu Ser Val 180 185 190 Ser Cys Trp Pro Leu Lys Asn Phe Asn Leu Leu Arg Leu Ala Thr Ile 195 200 205 Ser Ile Ala Val Leu Leu Thr Phe Ala Thr Leu Leu Leu Pro Phe Val 210 215 220 Leu Val Asp Gly Met Ser Gln Ile Gly Gln Ile Leu Phe Arg Val Phe 225 230 235 240 Pro Phe Ser Arg Gly Leu Phe Glu Asp Lys Val Ala Asn Phe Trp Cys 245 250 255 Thr Thr Asn Ile Leu Val Lys Tyr Lys Gln Leu Phe Thr Asp Lys Thr 260 265 270 Leu Thr Arg Ile Ser Leu Val Ala Thr Leu Ile Ala Ile Ser Pro Ser 275 280 285 Cys Phe Ile Ile Phe Thr His Pro Lys Lys Val Leu Leu Pro Trp Ala 290 295 300 Phe Ala Ala Cys Ser Trp Ala Phe Tyr Leu Phe Ser Phe Gln Val His 305 310 315 320 Glu Lys Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Glu Lys Asp Leu Asp Ile Ile Ser Met Val Cys Trp Ile Ser Asn Ile 340 345 350 Ala Phe Phe Ser Met Trp Pro Leu Leu Lys Arg Asp Gly Leu Ala Leu 355 360 365 Glu Tyr Phe Val Leu Gly Ile Leu Ser Asn Trp Leu Ile Gly Asn Leu 370 375 380 Asn Trp Ile Ser Lys Trp Leu Val Pro Ser Phe Leu Ile Pro Gly Pro 385 390 395 400 Thr Leu Ser Lys Lys Val Pro Lys Arg Asp Thr Lys Thr Val Val His 405 410 415 Thr His Trp Phe Trp Gly Ser Val Thr Phe Val Ser Tyr Leu Gly Ala 420 425 430 Thr Val Ile Gln Phe Val Asp Trp Leu Tyr Leu Pro Pro Ala Lys Tyr 435 440 445 Pro Asp Leu Trp Val Ile Leu Asn Thr Thr Leu Ser Phe Ala Cys Phe 450 455 460 Gly Leu Phe Trp Leu Trp Ile Asn Tyr Asn Leu Tyr Ile Leu 465 470 475 51836DNAKluyveromyces lactis 51atctctgttt caacagctct tgcattcatt ggttctttcg gtccaatcta tatctttgga 60ggatacaaga acttagtgca atcaatgcac aggatttttc catttgccag gggtatcttt 120gaagataaag ttgcgaattt ttggtgcgtt tctaatattt tcatcaaata tagaaatcta 180ttcactcaga aggatcttca attatactca ttactcgcaa cagttattgg gcttttacca 240tcattcatta taacattttt atacccgaag agacatttac taccatatgc tttggccgca 300tgttcgatgt cattcttctt attcagcttc caggttcatg aaaagacaat cttattacct 360ttacttccta ttacactctt gtacacgtca agagattgga atgttctatc attggtttgt 420tggattaaca acgtggcatt gtttacactc tggccattac tgaaaaagga caatctagta 480ttgcaatatg gagtcatgtt catgtttagc aattggttga tcggtaactt cagtttcgtc 540acaccacgct tcctcccaaa atttttgaca ccagggccat ccatcagtga tatagatgtt 600gattatagac gggcaagttt actacccaag agcctaatat ggagattaat cattgttggc 660tcatatattg caatggggat tattcatttt ctagactatt acgtctcccc gccatcaaaa 720taccctgatt tatgggtgct tgccaattgt tccttgggct tctcatgttt tgtgacattt 780tggatatgga acaattataa ttattcgaaa tgagaaacag cactttgcaa gattta 83652284PRTKluyveromyces lactisMOD_RES(116)..(127)Variable amino acid 52Ile Ser Val Ser Thr Ala Leu Ala Phe Ile Gly Ser Phe Gly Pro Ile 1 5 10 15 Tyr Ile Phe Gly Gly Tyr Lys Asn Leu Val Gln Ser Met His Arg Ile 20 25 30 Phe Pro Phe Ala Arg Gly Ile Phe Glu Asp Lys Val Ala Asn Phe Trp 35 40 45 Cys Val Ser Asn Ile Phe Ile Lys Tyr Arg Asn Leu Phe Thr Gln Lys 50 55 60 Asp Leu Gln Leu Tyr Ser Leu Leu Ala Thr Val Ile Gly Leu Leu Pro 65 70 75 80 Ser Phe Ile Ile Thr Phe Leu Tyr Pro Lys Arg His Leu Leu Pro Tyr 85 90 95 Ala Leu Ala Ala Cys Ser Met Ser Phe Phe Leu Phe Ser Phe Gln Val 100 105 110 His Glu Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 115 120 125 Thr Ser Arg Asp Trp Asn Val Leu Ser Leu Val Cys Trp Ile Asn Asn 130 135 140 Val Ala Leu Phe Thr Leu Trp Pro Leu Leu Lys Lys Asp Asn Leu Val 145 150 155 160 Leu Gln Tyr Gly Val Met Phe Met Phe Ser Asn Trp Leu Ile Gly Asn 165 170 175 Phe Ser Phe Val Thr Pro Arg Phe Leu Pro Lys Phe Leu Thr Pro Gly 180 185 190 Pro Ser Ile Ser Asp Ile Asp Val Asp Tyr Arg Arg Ala Ser Leu Leu 195 200 205 Pro Lys Ser Leu Ile Trp Arg Leu Ile Ile Val Gly Ser Tyr Ile Ala 210 215 220 Met Gly Ile Ile His Phe Leu Asp Tyr Tyr Val Ser Pro Pro Ser Gln 225 230 235 240 Glu Arg Tyr Lys Tyr Pro Asp Leu Trp Val Leu Ala Asn Cys Ser Leu 245 250 255 Gly Phe Ser Cys Phe Val Thr Phe Trp Ile Trp Asn Asn Tyr Xaa Leu 260 265 270 Phe Glu Arg Met Arg Asn Ser Thr Leu Gln Asp Leu 275 280 53284PRTKluyveromyces lactisMOD_RES(116)..(127)Variable amino acid 53Ile Ser Val Ser Thr Ala Leu Ala Phe Ile Gly Ser Phe Gly Pro Ile 1 5 10 15 Tyr Ile Phe Gly Gly Tyr Lys Asn Leu Val Gln Ser Met His Arg Ile 20 25 30 Phe Pro Phe Ala Arg Gly Ile Phe Glu Asp Lys Val Ala Asn Phe Trp 35 40 45 Cys Val Ser Asn Ile Phe Ile Lys Tyr Arg Asn Leu Phe Thr Gln Lys 50 55 60 Asp Leu Gln Leu Tyr Ser Leu Leu Ala Thr Val Ile Gly Leu Leu Pro 65 70 75 80 Ser Phe Ile Ile Thr Phe Leu Tyr Pro Lys Arg His Leu Leu Pro Tyr 85 90 95 Ala Leu Ala Ala Cys Ser Met Ser Phe Phe Leu Phe Ser Phe Gln Val 100 105 110 His Glu Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr 115 120 125 Thr Ser Arg Asp Trp Asn Val Leu Ser Leu Val Cys Trp Ile Asn Asn 130 135 140 Val Ala Leu Phe Thr Leu Trp Pro Leu Leu Lys Lys Asp Asn Leu Val 145 150 155 160 Leu Gln Tyr Gly Val Met Phe Met Phe Ser Asn Trp Leu Ile Gly Asn 165 170 175 Phe Ser Phe Val Thr Pro Arg Phe Leu Pro Lys Phe Leu Thr Pro Gly 180 185 190 Pro Ser Ile Ser Asp Ile Asp Val Asp Tyr Arg Arg Ala Ser Leu Leu 195 200 205 Pro Lys Ser Leu Ile Trp Arg Leu Ile Ile Val Gly Ser Tyr Ile Ala 210 215 220 Met Gly Ile Ile His Phe Leu Asp Tyr Tyr Val Ser Pro Pro Ser Gln 225 230 235 240 Glu Arg Tyr Lys Tyr Pro Asp Leu Trp Val Leu Ala Asn Cys Ser Leu 245 250 255 Gly Phe Ser Cys Phe Val Thr Phe Trp Ile Trp Asn Asn Tyr Xaa Leu 260 265 270 Phe Glu Arg Met Arg Asn Ser Thr Leu Gln Asp Leu 275 280 54238PRTKluyveromyces lactisMOD_RES(88)..(99)Variable amino acid 54Met His Arg Ile Phe Pro Phe Ala Arg Gly Ile Phe Glu Asp Lys Val 1 5 10 15 Ala Asn Phe Trp Cys Val Ser Asn Ile Phe Ile Lys Tyr Arg Asn Leu 20 25 30 Phe Thr Gln Lys Asp Leu Gln Leu Tyr Ser Leu Leu Ala Thr Val Ile 35 40 45 Gly Leu Leu Pro Ser Phe Ile Ile Thr Phe Leu Tyr Pro Lys Arg His 50 55 60 Leu Leu Pro Tyr Ala Leu Ala Ala Cys Ser Met Ser Phe Phe Leu Phe 65 70 75 80 Ser Phe Gln Val His Glu Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Tyr Thr Ser Arg Asp Trp Asn Val Leu Ser Leu Val Cys 100 105 110 Trp Ile Asn Asn Val Ala Leu Phe Thr Leu Trp Pro Leu Leu Lys Lys 115 120 125 Asp Asn Leu Val Leu Gln Tyr Gly Val Met Phe Met Phe Ser Asn Trp 130 135 140 Leu Ile Gly Asn Phe Ser Phe Val Thr Pro Arg Phe Leu Pro Lys Phe 145 150 155 160 Leu Thr Pro Gly Pro Ser Ile Ser Asp Ile Asp Val Asp Tyr Arg Arg 165 170 175 Ala Ser Leu Leu Pro Lys Ser Leu Ile Trp Arg Leu Ile Ile Val Gly 180 185 190 Ser Tyr Ile Ala Met Gly Ile Ile His Phe Leu Asp Tyr Tyr Val Ser 195 200 205 Pro Pro Ser Lys Tyr Pro Asp Leu Trp Val Leu Ala Asn Cys Ser Leu 210 215 220 Gly Phe Ser Cys Phe Val Thr Phe Trp Ile Trp Asn Asn Tyr 225 230 235 55252PRTKluyveromyces lactisMOD_RES(114)..(125)Variable amino acid 55Val Ser Thr Ala Leu Ala Phe Ile Gly Ser Phe Gly Pro Ile Tyr Ile 1 5 10 15 Phe Gly Gly Tyr Lys Asn Leu Val Gln Ser Met His Arg Ile Phe Pro 20 25 30 Phe Ala Arg Gly Ile Phe Glu Asp Lys Val Ala Asn Phe Trp Cys Val 35 40 45 Ser Asn Ile Phe Ile Lys Tyr Arg Asn Leu Phe Thr Gln Lys Asp Leu 50 55 60 Gln Leu Tyr Ser Leu Leu Ala Thr Val Ile Gly Leu Leu Pro Ser Phe 65 70 75 80 Ile Ile Thr Phe Leu Tyr Pro Lys Arg His Leu Leu Pro Tyr Ala Leu 85 90 95 Ala Ala Cys Ser Met Ser Phe Phe Leu Phe Ser Phe Gln Val His Glu 100 105 110 Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Thr Ser 115 120 125 Arg Asp Trp Asn Val Leu Ser Leu Val Cys Trp Ile Asn Asn Val Ala 130 135 140 Leu Phe Thr Leu Trp Pro Leu Leu Lys Lys Asp Asn Leu Val Leu Gln 145 150 155 160 Tyr Gly Val Met Phe Met Val Thr Pro Arg Phe

Leu Pro Lys Phe Leu 165 170 175 Thr Pro Gly Pro Ser Ile Ser Asp Ile Asp Val Asp Tyr Arg Arg Ala 180 185 190 Ser Leu Leu Pro Lys Ser Leu Ile Trp Arg Leu Ile Ile Val Gly Ser 195 200 205 Tyr Ile Ala Met Gly Ile Ile His Phe Leu Asp Tyr Tyr Val Ser Pro 210 215 220 Pro Ser Lys Tyr Pro Asp Leu Trp Val Leu Ala Asn Cys Ser Leu Gly 225 230 235 240 Phe Ser Cys Phe Val Thr Phe Trp Ile Trp Asn Asn 245 250 561617DNAMus musculus 56atgaagatga gacgctacaa gctctttctc atgttctgta tggctggcct gtgcctcata 60tccttcctgc acttctttaa gaccttatcc tatgtcacct tcccgagaga actggcctcc 120ctcagcccta acctcgtatc cagcttcttc tggaacaatg cccctgtcac tccccaggcc 180agtccggagc cgggtggccc cgacctattg cggacacccc tctactccca ctctcccctg 240ctccagccac tgtccccgag caaggccaca gaggaactgc accgggtgga cttcgtgttg 300ccggaggaca ccacggagta ttttgtgcgc accaaagctg gtggtgtgtg cttcaaacca 360ggtaccagga tgctggagaa accttcgcca gggcggacag aggagaagcc cgaagtgtct 420gagggctcct cagcccgggg acctgctcgg aggcccatga ggcacgtgtt gagtacgcgg 480gagcgcctgg gcagccgggg cactaggcgc aagtgggttg agtgtgtgtg cctgccaggc 540tggcacgggc ccagttgcgg ggtgcccacg gtggtgcagt attccaacct gcccaccaag 600gaacgcctgg tacccaggga ggtaccgagg cgggttatca acgccatcaa catcaaccac 660gagttcgacc tgctggatgt gcgcttccat gagctgggag atgttgtgga cgccttcgtg 720gtctgtgaat ctaatttcac cgcctacggg gagcctcggc cgctcaagtt ccgagagatg 780ctgaccaatg gcaccttcga gtacatccgc cacaaggtgc tctatgtctt cctggaccat 840ttcccacctg gtggccgtca ggacggctgg attgcggatg actacctgcg caccttcctc 900acccaggatg gcgtctcccg cctgcgcaac ctgcggcccg atgacgtctt tatcatcgac 960gatgcggacg agatccctgc gcgtgatggt gtgctgttcc tcaaactcta cgatggctgg 1020acagagccct tcgccttcca catgcggaag tccctgtatg gtttcttctg gaagcagccg 1080ggcacactgg aggtggtgtc aggctgcacc atggacatgc tgcaggccgt gtatgggctg 1140gatggcatcc gcctgcgccg ccgccagtac tacaccatgc ccaacttccg gcagtatgag 1200aaccgcaccg gccacatcct agtgcagtgg tctctcggca gccccctgca cttcgcgggc 1260tggcattgct cctggtgctt cacacccgag ggcatctact ttaaactcgt gtcagcccag 1320aatggcgact tcccccgctg gggtgactat gaggacaaga gggacctcaa ttacatccgc 1380agcttgatcc gcactggggg atggttcgac ggaacgcagc aggagtaccc tcctgcggac 1440cccagtgagc acatgtatgc tcctaaatac ctgctcaaga actatgacca gttccgctac 1500ttgctggaaa atccctaccg ggagcccaag agcactgtag agggtgggcg ccagaaccag 1560ggctcagatg gaaggccatc tgctgtcagg ggcaagttgg atacagtgga gggctag 1617572115DNAHomo sapiens 57gaaatgaacc tctcttattg atttttattg gcctagagcc aggagtactg cattcagttg 60actttcaggg taaaaagaaa acagtcctgg ttgttgtcat cataaacata tggaccagtg 120tgatggtgaa atgagatgag gctccgcaat ggaactgtag ccactgcttt agcatttatc 180acttccttcc ttactttgtc ttggtatact acatggcaaa atgggaaaga aaaactgatt 240gcttatcaac gagaattcct tgctttgaaa gaacgtcttc gaatagctga acacagaatc 300tcacagcgct cttctgaatt aaatacgatt gtgcaacagt tcaagcgtgt aggagcagaa 360acaaatggaa gtaaggatgc gttgaataag ttttcagata ataccctaaa gctgttaaag 420gagttaacaa gcaaaaaatc tcttcaagtg ccaagtattt attatcattt gcctcattta 480ttgaaaaatg aaggaagtct tcaacctgct gtacagattg gcaacggaag aacaggagtt 540tcaatagtca tgggcattcc cacagtgaag agagaagtta aatcttacct catagaaact 600cttcattccc ttattgataa cctgtatcct gaagagaagt tggactgtgt tatagtagtc 660ttcataggag agacagatat tgattatgta catggtgttg tagccaacct ggagaaagaa 720ttttctaaag aaatcagttc tggcttggtg gaagtcatat caccccctga aagctattat 780cctgacttga caaacctaaa ggagacattt ggagactcca aagaaagagt aagatggaga 840acaaagcaaa acctagatta ctgttttcta atgatgtatg ctcaagaaaa gggcatatat 900tacattcagc ttgaagatga tattattgtc aaacaaaatt attttaatac cataaaaaat 960tttgcacttc aactttcttc tgaggaatgg atgattctag agttttccca gctgggcttc 1020attggtaaaa tgtttcaagc gccggatctt actctgattg tagaattcat attcatgttt 1080tacaaggaga aacccattga ttggctcctg gaccatattc tctgggtgaa agtctgcaac 1140cctgaaaaag atgcaaaaca ttgtgataga cagaaagcaa atctgcgaat tcgcttcaga 1200ccttcccttt tccaacatgt tggtctgcac tcatcactat caggaaaaat ccaaaaactc 1260acggataaag attatatgaa accattactt cttaaaatcc atgtaaaccc acctgcggag 1320gtatctactt ccttgaaggt ctaccaaggg catacgctgg agaaaactta catgggagag 1380gatttcttct gggctatcac accgatagct ggagactaca tcttgtttaa atttgataaa 1440ccagtcaatg tagaaagtta tttgttccat agcggcaacc aagaacatcc tggagatatt 1500ctgctaaaca caactgtgga agttttgcct tttaagagtg aaggtttgga aataagcaaa 1560gaaaccaaag acaaacgatt agaagatggc tatttcagaa taggaaaatt tgagaatggt 1620gttgcagaag gaatggtgga tccaagtctc aatcccattt cagcctttcg actttcagtt 1680attcagaatt ctgctgtttg ggccattctt aatgagattc atattaaaaa agccaccaac 1740tgatcatctg agaaaccaac acattttttc ctgtgaattt gttaattaaa gatagttaag 1800catgtatctt ttttttattt ctacttgaac actacctctt gtgaagtcta ctgtagataa 1860gacgattgtc atttccactt ggaaagtgaa tctcccataa taattgtatt tgtttgaaac 1920taagctgtcc tcagatttta acttgactca aacatttttc aattatgaca gcctgttaat 1980atgacttgta ctattttggt attatactaa tacataagag ttgtacatat tgttacattc 2040tttaaatttg agaaaaacta atgttacata cattttatga agggggtact tttgaggttc 2100acttatttta ctatt 2115583226DNAMus musculus 58attgctagag agagatggct ttcttttctc cctggaagtt gtcctctcag aagctgggct 60ttttcctggt gactttcggc ttcatctggg gcatgatgct tctgcacttc accatccagc 120agcggactca gcccgagagc agctccatgt tacgggagca gatccttgac ctcagcaaga 180ggtacattaa ggcactggca gaggagaaca gggacgtggt ggatggcccc tacgctggtg 240tcatgacagc ctatgatctg aagaaaacgc tcgccgtctt gctggataac atcctgcagc 300gcattggcaa gctcgagtca aaggtggaca atctggtcaa cggcacagga gcgaactcca 360ccaactccac cacggctgtc cccagcttgg tgtcgcttga gaaaattaat gtggcagata 420tcattaatgg agttcaggaa aaatgtgtat tgcctcctat ggatggctac ccccactgcg 480aggggaaaat caagtggatg aaggacatgt ggcgctcgga cccctgctac gcagactatg 540gagtggacgg gacctcctgc tcctttttta tttacctcag tgaggttgaa aattggtgtc 600ctcgtttacc ttggagagca aaaaatccct atgaagaagc tgatcataac tcattggcgg 660aaatccgtac ggattttaac attctctacg gcatgatgaa gaagcacgag gagttccgtt 720ggatgaggct tcggatccgg cgaatggctg acgcgtggat ccaagctatc aagtctctgg 780cggagaaaca aaaccttgag aagaggaaac ggaagaaaat ccttgttcac ctggggctcc 840tgaccaagga atcgggcttc aagattgcgg agacagcatt cagcggtggc cctctgggtg 900aactcgttca gtggagtgac ttaatcacat ctctgtacct gctgggccat gacatccgga 960tctcggcctc actggctgag ctcaaggaga taatgaagaa ggttgttgga aaccggtctg 1020gctgtccaac tgtaggagac agaatcgttg agctgattta tatcgatatt gtgggacttg 1080ctcaatttaa gaaaacacta gggccatcct gggttcatta ccagtgcatg ctccgggtgc 1140tagactcctt tggaacagaa cctgagttca atcatgcgag ctatgcccag tcaaaaggcc 1200acaagacccc ctggggaaag tggaatctga acccgcagca gttttacacc atgttccctc 1260ataccccaga caacagcttt ctgggcttcg tggtggagca gcacctgaac tccagcgaca 1320ttcaccacat caacgagatc aaaaggcaga accagtccct tgtgtatggc aaagtggata 1380gtttctggaa gaataagaaa atctacctgg atatcattca cacgtacatg gaagtgcacg 1440ccactgttta tggctccagt accaagaaca ttcccagtta cgtgaaaaac catggcattc 1500tcagtggacg tgacctgcag tttcttctcc gggaaaccaa gctgttcgtt gggctcggat 1560tcccttatga aggcccagct cccctggagg ccatcgcgaa tggatgtgct ttcctgaacc 1620ccaagttcaa ccctcccaaa agcagcaaaa acacagactt cttcattggc aagccaacac 1680tgagagagct gacatcccag catccttacg cagaagtctt catcggccgg ccacacgtct 1740ggactgtgga tctcaataac cgagaggaag tagaagatgc agtaaaagcc atcttaaacc 1800agaagattga gccgtatatg ccatatgagt tcacatgtga aggcatgctg cagagaatca 1860acgctttcat tgaaaaacag gacttctgcc atggccaagt gatgtggccg cccctcagcg 1920ccctgcaggt taagctggct gagccagggc agtcctgcaa acaggtgtgc caggagagcc 1980agctcatctg cgagccatcc ttctttcaac acctcaacaa ggaaaaggac ctgctgaagt 2040ataaggtgac ctgccaaagc tcagaactgt acaaggacat cctggtgccc tccttctacc 2100ccaagagcaa gcactgtgtg ttccaagggg acctcctgct cttcagttgt gccggagccc 2160atcccacaca ccagcggatc tgcccctgcc gggacttcat caagggccaa gtggccctct 2220gcaaagactg cctatagcat cgctgccctg aattaactca gacgggaaag acgtggctcc 2280actgggcagg gccaaggggc acaaagacat tcagggactc tgaccagagc ctgagatctt 2340tggtccaggg cttgagttta gtaccgctcc agccacagcc agtgcatccc agtttacacc 2400aaaaccacaa gggaacaggt tagaacagga acctgggttc tcctcagtgt aaggaatgtc 2460ctctctgtct gggagatcga gcgactgtag ggaaaggatc caggcagttg ctcccgggaa 2520tttttttttt tttttttttt aaagaaggga taaaagtccg gagactcatt caaactgaaa 2580acaaaacagg aagagggaat tgagccaatt gggaaggact ttggggccga tcctaaacca 2640attaatttat ttatttggga ggatgggggc gggctcggga gggaggagag gggttgaaca 2700gtttcctttt gttcctcact gttaattcgc ccaccttcgg gcccttcttg ttctgcagcg 2760ccaagcaggg tgcagagggg ctgtggcttg cttgaggggc cactgtgggg cttcactcct 2820ggtcacaggt ggcagcagag aaaagagatg tctataagca gggggatgta gctcagtttg 2880tagaatgctt gcatagcata aatgaagtcc tgggttccat ccccagcacc acataaatgc 2940aggtaagaaa cagagtcagg aggaccaagc attctccttg gctacataac aaaagcaagg 3000cctttgtccc catgtcttgg ctacaagaga ccctatctca gaaaattgtg ggggggaggg 3060ggggggaaat ggccttgaaa acacagccag tcactgtcac tgcattgcca gaactggtgg 3120atcccaggtg tgcttggcag ataacagcta aaaggcacat aaccttggtg gggaaataaa 3180tgcctgtggt gtcctgaggg ccccaccaag ttccaaaaaa aaaaaa 3226

Claims

1-60. (canceled)

61. A Pichia pastoris host cell wherein the host cell lacks OCH1 activity and has diminished or depleted activity of one or more enzymes selected from the group consisting of: (a) an enzyme having dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity; (b) an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity; and (c) an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity.

62. The host cell of claim 61, wherein the diminished or depleted enzyme has dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity.

63. The host cell of claim 61, herein the host cell has depleted dolichyl-P-Man:Man5 GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase activity.

64. The host cell of claim 62 or 63, further expressing: (i) an a1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and (iii) a recombinant glycoprotein.

65. The host cell of claim 64, further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures.

66. The host cell of claim 65, further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI.

67. The host cell of claim 64, further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.

68. The host cell of claim 60, wherein the host cell has diminished or depleted activity of an enzyme having dolichyl-P-Man:Man6GlcNAc2-PP-dolichyl alpha-1,2 mannosyltransferase activity.

69. The host cell of claim 62 or 63, further expressing one or more nucleic acids encoding: (i) an .alpha.1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and (iii) a recombinant glycoprotein.

70. The host cell of claim 69, further expressing a nucleic acid molecule encoding an .alpha.-1,3 and/or .alpha.-1,2-1,3 mannosidase catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell.

71. The host cell of claim 70, further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures.

72. The host cell of claim 71, further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI.

73. The host cell of claim 69, further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.

74. The host cell of claim 60, wherein the host cell has diminished or depleted activity of an enzyme having dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl alpha-1,6 mannosyltransferase activity.

75. The host cell of claim 73, further expressing one or more nucleic acid molecules encoding: (i) an .alpha.1,2-mannosidase catalytic domain fused to a targeting peptide that targets the endoplasmic reticulum (ER) or Golgi apparatus in the host cell, (ii) a GlcNAc transferase I (GnT I) catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell, and (iii) a recombinant glycoprotein.

76. The host cell of claim 75, further expressing a nucleic acid molecule 1,2-1,3 mannosidase catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell.

77. The host cell of claim 74, further expressing a nucleic acid molecule encoding a GnT II catalytic domain fused to a targeting peptide that targets the ER or Golgi apparatus of the host cell and the method results in the production within the host cell of recombinant glycoproteins having N-glycans attached thereto comprising GlcNAc2Man3GlcNAc2 core structures.

78. The host cell of claim 77, further expressing one or more nucleic acid molecules encoding one or more enzyme activities selected from galactosyltransferase, sialyltransferase, fucosyltransferase, and GlcNAc transferase III, IV, V, and VI.

79. The host cell of claim 75, further expressing one or more nucleic acid molecules encoding one or more sugar transporters selected from UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter, and CMP-sialic acid transporter.