Expression of Functional Soluble Forms of Human β-1,4-Galactosyltransferase I, α-2,6-Sialyltransferase, and α-1,3-Fucosyltransferase VI in the Methylotrophic Yeast Pichia pastoris

Malissard, Martine; Zeng, Steffen; Berger, Eric G.

doi:10.1006/bbrc.1999.1946

Cited by 41 publications

(14 citation statements)

References 29 publications

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“…Attempts have been made to engineer glycosylation pathways so as to enrich for specific glycoforms in mAbs secreted by a variety of expression systems, including cell lines from mammalian species (Davies et al, 2001), yeast (Bobrowicz et al, 2004;Vervecken et al, 2004), and plants (Ko et al, 2003;Shriver et al, 2004;Tekoah et al, 2004). An emerging alternative is the use of enzymes postproduction to remodel glycan chains, a process made possible by high-level overexpression of glycosyltransferases in soluble form (Malissard et al, 2000;Perugino et al, 2004). We have demonstrated previously the preparative in vitro remodeling of glycan chains on a therapeutic glycoprotein at the 10 g scale, including enzymatic sialylation and fucosylation of complex biantennary N-glycans to homogeneous sialyl Lewis X (sLe x )-active glycoforms (Thomas et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

Warnock

Bai

Autote

et al. 2005

Biotech & Bioengineering

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The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.

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Section: Discussionmentioning

confidence: 99%

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

Warnock

Bai

Autote

et al. 2005

Biotech & Bioengineering

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“…In terms of large production of soluble ST6Gal I, Malissard et al have expressed the enzyme in yeast cells [54]. Although yield (0.3 units/liter) of the enzyme expressed in yeast is twice higher than that expressed in E. coli, the enzyme produced in bacteria has some merits to apply for generation of artificial carbohydrate molecules.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

et al. 2005

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A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13 degrees C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac alpha 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates.

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“…Several successful efforts at heterologous expression in alternative hosts, such as Escherichia coli, 21 23) fungi 24,25) and insect cells, 26,27) have been reported. However, only a few enzymes have been expressed on an industrial scale.…”

Section: Expression Of Glycosyltransferases In Methylotrophic Yeast Cmentioning

confidence: 99%

Expression System for Human Glycosyltransferases and Its Application

Chiba¹,

Ito²,

Sato³

et al. 2010

J. Appl. Glycosci.

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Glycosylation of proteins and lipids is the most complex of their various co translational and post translational modifications. Glycan modification contributes to the folding and conformational stability of many proteins, 1) and modified glycoproteins and glycolipids on the cell surface are known to act as ligands for host pathogen interactions, 2) cell adhesion and cell signaling.3) These phenomena are influenced by changes in glycan structures. Since it is known that glycans on proteins and lipids are synthesized by sequential reactions by glycosyltransferases, the alteration of the glycan structure is dependent on the expression of glycan synthesis related genes encoding glycosyltransferases, sulfotransferases, glycosidases, sugar nucleotide synthetases, and sugar nucleotide transporters. Glycan structures are defined by not only the expression level of the genes but also several other factors, such as substrate specificity, competition of more than two glycosyltransferases for the same substrate, localization of glycosyltransferases, and concentration of donor substrate as sugar nucleotides in the Golgi apparatus. Therefore, the expression of glycosyltransferases and the determination of their enzymatic characterizations in detail are significant for the elucidation of the biosynthetic machinery of glycans.In addition to basic study of glycosyltransferases, recombinant enzymes are available for the synthesis of glycans and glycopeptides and for the modification of glycoproteins in vitro. In recent years, mass spectrometry, 4,5) high performance liquid chromatography (HPLC), 6,7) and lectin microarray systems 8,9) have been developed for the structural analysis of glycans. A glycan library composed of a variety of structure defined glycans is indispensable for quantitative analyses using these technologies. Since human glycosyltransferases have a strict specificity toward donor and acceptor substrates, it is reasonable to employ in vitro synthesis of glycans by glycosyltransferases for producing glycan standards. Glycan array technology, and the ease with which the glycans can be customized for display, may enhance our ability to detect unknown lectins in mammals and to probe pathogen specific glycan interactions. 10,11) Application of glycosyltransferases in other areas of interest includes the modification of glycans attached to glycoproteins, which has also become an important topic in recent years. Because biologics and biosimilar drugs such as therapeutic antibodies and cytokines are the largest class of new drug candidates being developed by pharmaceutical companies, there are strong concerns that the heterogeneity of the glycan structure may sometimes affect their in vivo activity. Therefore, the development of technology to produce glycoproteins with homogeneous glycans is essential.In our research center, the Glycogene (GG) Project was started in 2001, in which as many novel genes as possible, which were the candidates for glycogenes including glycosyltransferases and sugar nucleotide transporters, ...

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Expression of Functional Soluble Forms of Human β-1,4-Galactosyltransferase I, α-2,6-Sialyltransferase, and α-1,3-Fucosyltransferase VI in the Methylotrophic Yeast Pichia pastoris

Cited by 41 publications

References 29 publications

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

Expression System for Human Glycosyltransferases and Its Application

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