Mannosidases capable of decapping mannose-1-phosphate-6-mannose linkages and demannosylating phosphorylated n-glycans and methods of promoting mammalian cell uptake of glycoproteins
A kind of mannosidase, mannosyl technology, applied in the field of mannosidase, can solve problems such as disadvantage
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Embodiment 1
[0142] Generation of human alpha glucosidase expression strain
[0143] Yarrowia lipolytica strain OXYY1589 was constructed as follows and contained three copies of the human alpha glucosidase gene (huGAAA, also known as acid alpha glucosidase or acid maltase EC3.2.1.3) and two copies of lipolytic Yarrowia MNN4 gene. The genotype of strain OXY1589 is as follows:
[0144] MatA, leu2-958, ura3-302, xpr2-322,
[0145] gut2-744, ade2-844
[0146] POX2-Lip2pre-huGAA:URA3Ex::zeta
[0147] POX2-Lip2pre-huGAA:LEU2Ex::zeta
[0148] POX2-Lip2pre-hGM-CSF:GUTEx::zeta
[0149] YlMNN4-POX2-hp4d-YLMNN4:ADE2::PT targeting
[0150] All transformations were performed according to well-established protocols with modifications for the different selectable markers. Unless otherwise specified, huGAA integration fragments were obtained by NotI restriction digestion of expression plasmids to remove the kanamycin resistance gene from the expression plasmids. Fragments derived from the restrict...
Embodiment 2
[0166] Fed-batch culture of strain OXYY1589
[0167] For the production of huGAA from strain OXYY1589 (Example 1), a fed-batch process was set up using a 10 L stirred tank with a working volume of 6-8 liters. The process is divided into two phases:
[0168] 1) Batch growth on glucose to form biomass
[0169] 2) By inducing the formation of the product with the aid of limited oleic acid feed
[0170] Typically, the batch stage is about 20 hours (h) and the production stage is about 72 hours. At the end of the process, the culture broth was centrifuged and the supernatant collected. The supernatant was used as starting material to purify huGAA (see Example 3).
[0171] The following parameters were controlled during fermentation. Aeration was maintained at a constant value of 1.5 vvm air (volume per minute). Initially, dissolved oxygen (DO) was maintained at 30%. Depending on the DO level, the agitation was increased from 600 to 1200 rpm. Once it reaches a maximum of 120...
Embodiment 3
[0174] Purification of recombinant huGAA (rhGAA)
[0175] The post-incubation supernatant (see Example 2) was clarified via depth filtration. The resulting material was then concentrated 20-fold via tangential flow filtration (TFF) and diafiltered against 20 mM sodium phosphate pH 6 and 100 mM NaCl using a 10 kDa MWCO membrane (Millipore).
[0176] Purification of rhGAA was initiated by adding ammonium sulfate up to a concentration of 1M. After centrifugation, the supernatant was loaded on a Toyopearl-Phenyl 650M (Tosoh Biosciences) XK16 / 40 packed column. A linear gradient of 1 to 0 M ammonium sulfate was applied for elution. Those fractions containing rhGAA were then pooled and buffer exchanged into 10 mM BIS-TRIS pH 6. Further purification was achieved via anion exchange chromatography on a Source30Q Tricorn 10 / 50 or XK25 / 20 packed column (GE Healthcare) using a linear salt gradient from 0 to 1 M NaCl. The resulting GAA-containing fractions were then concentrated before ...
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