Protein glycosylation modification in Pichia pastoris

Details Protein glycosylation modification in Pichia pastoris Protein glycosylation modification in Pichia pastoris

2001-06-29

2004-10-12

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S254220, C435S254230, C435S320100, C435S483000

Reexamination Certificate

active

06803225

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods and vectors useful for genetically modifying the glycosylation process in methylotrophic yeast strains for the purpose of producing glycoproteins with reduced glycosylation. The present invention further relates to methylotrophic yeast strains generated using the present methods and vectors, as well as glycoproteins produced from such genetically modified strains.
BACKGROUND OF THE INVENTION
The methylotrophic yeasts including
Pichia pastoris
have been widely used for production of recombinant proteins of commercial or medical importance. However, production and medical applications of some therapeutic glycoproteins can be hampered by the differences in the protein-linked carbohydrate biosynthesis between these yeasts and the target organism such as a mammalian subject.
Protein N-glycosylation originates in the endoplasmic reticulum (ER), where an N-linked oligosaccharide (Glc
3
Man
9
GlcNAc
2
) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate Asn of a nascent protein. This is an event common to all eukaryotic N-linked glycoproteins. The three glucose residues and one specific &agr;-1,2-linked mannose residue are removed by specific glucosidases and an (&agr;-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man
8
GlcNAc
2
. The protein with this core sugar structure is transported to the Golgi apparatus where the sugar moiety undergoes various modifications. There are significant differences in the modifications of the sugar chain in the Golgi apparatus between yeast and higher eukaryotes.
In mammalian cells, the modification of the sugar chain proceeds via 3 different pathways depending on the protein moiety to which it is added. That is, (1) the core sugar chain does not change; (2) the core sugar chain is changed by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removing the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; or (3) the core sugar chain is first converted into Man
5
GlcNAc
2
by removing 3 mannose residues with mannosidase I; Man
5
GlcNAc
2
is further modified by adding GlcNAc and removing 2 more mannose residues, followed by sequentially adding GlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld,
Ann. Rev. Biochem.
54: 631-664, 1985; Chiba et al
J. Biol. Chem.
273: 26298-26304, 1998).
In yeast, the modification of the sugar chain in the Golgi involves a series of additions of mannose residues by different mannosyltransferases (“outer chain” glycosylation). The structure of the outer chain glycosylation is specific to the organisms, typically with more than 50 mannose residues in
S. cerevisiae
, and most commonly with structures smaller than Man
14
GlcNAc
2
in
Pichia pastoris
. This yeast-specific outer chain glycosylation of the high mannose type is also denoted hyperglycosylation.
Hyperglycosylation is often undesired since it leads to heterogeneity of a recombinant protein product in both carbohydrate composition and molecular weight, which may complicate the protein purification. The specific activity (units/weight) of hyperglycosylated enzymes may be lowered by the increased portion of carbohydrate. In addition, the outer chain glycosylation is strongly immunogenic which is undesirable in a therapeutic application. Moreover, the large outer chain sugar can mask the immunogenic determinants of a therapeutic protein. For example, the influenza neuraminidase (NA) expressed in
P. pastoris
is glycosylated with N-glycans containing up to 30-40 mannose residues. The hyperglycosylated NA has a reduced immunogenicity in mice, as the variable and immunodominant surface loops on top of the NA molecule are masked by the N-glycans (Martinet et al.
Eur J. Biochem.
247: 332-338, 1997).
Therefore, it is desirable to genetically engineer methylotrophic yeast strains in which glycosylation of proteins can be manipulated and from which recombinant proteins can be produced that would not be compromised in structure or function by large N-glycan side chains.
SUMMARY OF THE INVENTION
The present invention is directed to methods and vectors useful for genetically modifying the glycosylation process in methylotrophic yeast strains to produce glycoproteins with reduced glycosylation. Methylotrophic yeast strains generated using the present methods and vectors, as well as glycoproteins produced from such genetically modified strains are also provided.
In one embodiment, the present invention provides vectors useful for making genetically engineered methylotrophic yeast strains which are capable of producing glycoproteins with reduced glycosylation.
In one aspect, the present invention provides “knock-in” vectors which are capable of expressing in a methylotrophic yeast strain one or more proteins whose enzymatic activities lead to a reduction of glycosylation in glycoproteins produced by the methylotrophic yeast strain.
In a preferred embodiment, the knock-in vectors of the present invention include a nucleotide sequence coding for an &agr;-1,2-mannosidase or a functional part thereof and are capable of expressing the &agr;-1,2-mannosidase or the functional part in a methylotrophic yeast strain. A preferred nucleotide sequence is a nucleotide sequence encoding the &agr;-1,2-mannosidase of a fungal species, and more preferably,
Trichoderma reesei
. Preferably, the &agr;-1,2-mannosidase expression vector is engineered such that the &agr;-1,2-mannosidase or a functional part thereof expressed from the vector includes an ER-retention signal. A preferred ER-retention signal is HDEL (SEQ ID NO: 1). The &agr;-1,2-mannosidase coding sequence can be operable linked to a constitutive or inducible promoter, and a 3′ termination sequence. The vectors can be integrative vectors or replicative vectors. Particularly preferred &agr;-1,2-mannosidase expression vectors include pGAPZMFManHDEL, pGAPZMFManMycHDEL, pPICZBMFManMycHDEL, pGAPZmManHDEL, pGAPZmMycManHDEL, pPIC9mMycManHDEL and pGAPZmMycManHDEL.
In another preferred embodiment, the knock-in vectors of the present invention include a sequence coding for a glucosidase II or a functional part thereof and are capable of expressing the glucosidase II or the functional part in a methylotrophic yeast strain. A preferred nucleotide sequence is a nucleotide sequence encoding the glucosidase II of a fungal species, and more preferably,
Saccharomyces cerevisiae
. Preferably, the glucosidase II expression vector is engineered such that the glucosidase II or a functional part thereof expressed from the vector includes an ER-retention signal. A preferred ER-retention signal is HDEL (SEQ ID NO: 1). The glucosidase II coding sequence can be operable linked to a constitutive or inducible promoter, and a 3′ termination sequence. The vectors can be integrative vectors or replicative vectors. Particularly preferred glucosidase II expression vectors include pGAPZAGLSII, pPICZAGLSII, pAOX2ZAGLSII, pYPTIZAGLSII, pGAPADEglsII, pPICADEglsII, pAOX2ADEglsII, pYPTIADEglsII, pGAPZAglsIIHDEL and pGAPADEglsIIHDEL.
Expression vectors which include both of an &agr;-1,2-mannosidase expression unit and a glucosidase II expression unit are also provided by the present invention.
In another aspect, the present invention provides “knock-out” vectors which, when introduced into a methylotrophic yeast strain, inactivate or disrupt a gene thereby facilitating the reduction in the glycosylation of glycoproteins produced in the methylotrophic yeast strain.
In one embodiment, the present invention provides a “knock-out” vector which, when introduced into a methylotrophic yeast strain, inactivates or disrupts the Och1 gene. A preferred Och1 knock-out vector is pBLURA5′PpOCH1.
Still another embodiment of the present invention provides vectors which include both a knock-in unit and

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