O-linked glycosylation using n-acetylglucosaminyl transferases
A technology of glycosylation and transferase, applied in the direction of transferase, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as inappropriateness and reduced biological activity of polypeptides
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[0663] Preparation of Modified Sugars
[0664] Typically, a covalent bond is formed between the sugar moiety and the modifying group through the use of a reactive functional group, which is converted into a new organic functional group or a non-reactive species, usually through a linkage process. To form this bond, the modifying group and the sugar moiety carry complementary reactive functional groups. The reactive functional group can be located anywhere on the sugar moiety.
[0665] Reactive groups and reactive classes useful in the practice of the present invention are generally those well known in the art of bioconjugate chemistry. A presently advantageous class of reactions available with reactive sugar moieties are those that proceed under relatively mild conditions. These reactions include, but are not limited to, nucleophilic substitution (e.g., reaction of amines and alcohols with acid halides, active esters), electrophilic substitution (e.g., enamine reaction), and...
Embodiment 1
[0994] Preparation of Mutant Interferon-α-2b-GlcNH-Glycine-PEG-30kDa
[0995] Mutant IFN-α-2b (30 mg, 1.55 micromolar) was buffer exchanged into reaction buffer (50 mM Tris, MgCl) using Centrieon Plus-20 centrifugal filters (5 kDa MWCO) 2 , pH 7.8), to a final protein concentration of 10 mg / mL. Then, UDP-GlcNH-glycine-PEG-30kDa (2 molar equivalents) and MBP-GlcNAc transferase (20mU / mg protein) were added. The reaction mixture was incubated at 32°C until the reaction was complete. The extent of the reaction was determined by SDS-PAGE gel. The product, IFN-a-2b-GlcNH-glycine-PEG-30kDa, was purified as described in the literature (SP-sepharose and Superdex 200 chromatography) before formulation.
[0996] IFNα mutant :
[0997] MCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVPVS 106 RAPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE
[0998] (SEQ ID NO: 234)
[0999] UDP-GlcNH-Glycine-PEG-30kDa :
[1000] ...
Embodiment 2
[1002] Preparation of mutant interferon-α-2b-GlcNH-hexanoylamido-PEG-40kDa
[1003] Mutant IFN-α-2b (1 mg) was buffer exchanged into reaction buffer (50 mM HEPES, MgCl 2 , pH 7.4, 100 mM NaCl), to a final protein concentration of 1 mg / mL. Then, UDP-GlcNH-hexanoylamido-PEG-40 kDa (2 molar equivalents) and MBP-GlcNAc transferase (100 mU / mg protein) were added. The reaction mixture was incubated at 32°C until the reaction was complete. The extent of the reaction was determined by SDS-PAGE gel. The product, IFN-[alpha]-2b-GlcNH-hexanoylamido-PEG-40kDa, was purified as described in the literature (SP-sepharose and Superdex 200 chromatography) before formulation.
[1004] IFNα mutant :
[1005] MCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGPV 106 SRPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE
[1006] (SEQ ID NO: 235)
[1007] UDP-GlcNH-hexanoylamido-PEG-40kDa :
[1008]
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