Enzyme assay of polypeptide N-acetylgalactosaminyltransferase, β1,3-glycosyltransferase, and β1,4-glycosyltransferases. [5] Recombinant glycosyltransferase production in HEK293T cells using GGENTRtr library.
Human embryonic kidney (HEK) 293T cells (American type cell collection)
pFLAG-CMV3 (Sigma Aldrich, St. Louis, MO)
Gateway vector conversion system (Invitrogen/Life Technology, Carlsbad, CA)
Gateway LR clonase enzyme mix (Invitrogen/Life Technology)
Lipofectamine 2000 (Invitrogen/Life Technology)
Opti-MEM I (Invitrogen/Life Technology)
Anti-FLAG M2 agarose affinity gel (Sigma Aldrich, St. Louis, MO)
TBS (Tris-buffered saline)
FLAG peptide (Sigma Aldrich)
3X FLAG peptide (Sigma Aldrich)
CO2 incubator
Construction of glycosyltransferase expression vector
In this section, pFLAG-CMV3-DEST vector is chosen as a gateway destination vector for enzyme expression and gateway LR clonase enzyme mix is used for LR recombination. The destination vector can be introduced from your favorite vectors by using gateway vector conversion systems.
Mix 1 μL of GGENTRtr vector, 1 μL of destination vector (pFLAG-CMV3-DEST), 1 μL of 5X LR Clonase reaction buffer, 1 μL of TE, and 1 μL of LR Clonase and incubate at 25℃ for 1 h.
Add 1 μL of the Proteinase K to terminate the reactions and incubate at 37℃ for 10 min.
Transform 1 μL of LR reaction into DH5α competent cells and transformants are selected on LB plates containing 100 μg/mL ampicillin.
Recombinant enzyme preparation
When a pFLAG-CMV3 plasmid containing catalytic region of each glycosyltransferase is transfected into HEK293T, the recombinant enzyme can be prepared from culture medium.
TRANSFECTION
One day before transfection, seed 2.0 × 106 cells in 10 mL of DMEM (with 10% FCS, without antibiotics) on a 10 cm dish.
Dilute 30 μg of plasmid DNA in 1.5 mL of Opti-MEM I and mix gently.
Dilute 30 μL of LipofectAmine 2000 in 1.5 mL of Opti-MEM I and mix gently. Incubate the mixture for 5 min at room temperature.
Mix the dilute DNA solution with the diluted Lipofectamine 2000 solution. Mix gently and incubate for 20 min at room temperature.
Drop the mixed solution on confluent HEK293T cells. Mix gently and incubate for 48–72 h at 37°C in a CO2 incubator.
Collect the culture medium and remove the cell debris by centrifugation.
AFFINITY PURIFICATION
Mix the medium with anti-FLAG M2 agarose affinity gel and incubate for O/N at 4°C with gentle agitation by a rotary shaker.
Wash the enzyme-agarose gel mixture three times with TBS.
The enzyme-agarose gel suspension can be directly used for the enzymatic reactions of glcosyltransferase. When a free recombinant enzyme is required, it can be eluted from agarose gel by competition with FLAG or 3X FLAG peptide.
GGENTRtr library contains catalytic domain of more than 150 of human glycogenes including glycosyltransferases and sulfotransferases as Entryclone of gateway system. When you produce recombinant enzymes of glycosyltransferase and sulfotransferase in your laboratory, GGENTRtr library will be very useful resource to do it in your favorite expression system such as mammalian, insect, and yeast cells depending on your destination vectors. GGENTRtr library is available from NITE biological resource center (NBRC, http://www.nbrc.nite.go.jp). The detail of each Entryclone such as gene name, position cloned, fusion frame, vector, sequence, etc is able to refer in glycogene database (GGDB, http://riodb.ibase.aist.go.jp/rcmg/ggdb/). GGENTRtr library was constructed in the “glycogene (GG) project” of New Energy and Industrial Technology Development Organization (NEDO).
