Yeast alpha-1,6-mannosyltransferase (Och1p) is yeast specific mannosyl transferase which transfer the first mannose for synthesizing yeast huge mannose outer chain in N-glycans. In Saccaromyces cerevisiae, huge mannose outer chain consists of 50–100 mannose residues, but disruption of OCH1 gene could remove almost huge outer mannose chain from N-linked oligosaccharide. Therefore, disruption of Och1p is very important for construct useful host yeast strain for production mammalian glycoproteins. This protocol for enzyme assay of Och1p is suitable for S. cerevisiae’s Och1p, but another yeast Och1p may be assayed using this protocol. |
Category | Glycosyltransferases & related proteins |
Protocol Name | Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p) |
Authors
|
Nakayama, Ken-ichi
Biomass Treatment Group, Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST)
|
KeyWords |
|
Reagents
|
● |
Yeast cells (OCH1 wild type) |
● |
Yeast Extract (BD, Franklin Lakes, NJ) |
● |
|
● |
Glucose (Wako Pure Chemical Industries Ltd., Osaka, Japan) |
● |
50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol and 2 μg/mL each of proteinase inhibitor (antipain, chymostatin, leupeptin and pepstatin A) |
● |
Glass beads (0.45–0.50 ø mm) |
● |
GDP-mannose (Sigma-Aldrich, St. Louis, MO) |
● |
Man8GlcNAc2-PA (Takara Bio Inc., Otsu, Japan) |
● |
Asahipak NH2P-50 (0.46 × 25 cm) (Showa Denko, K.K., Tokyo, Japan) |
● |
200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 3 : 7 |
● |
200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 7 : 3 |
|
Instruments
|
● |
|
● |
Homogenizer (B.Braun or Vortex mixer) |
● |
|
● |
|
● |
|
● |
|
|
Methods |
1. |
Preparation of yeast membranes and alpha-1,6-mannosyltransferase assay
|
1) |
Yeast cells are grown in 250 mL of YPAD medium at 30˚C for overnight. |
Comment 0
|
|
2) |
Collect cells by centrifugation at 3000 × g for 5 min and wash with 1% KCl. |
Comment 0
|
|
3) |
Resuspend cells in 5 mL of 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol and 2 mg/mL each of proteinase inhibitor. |
Comment 0
|
|
4) |
Add glass beads (0.45–0.50 ø mm) to half of cell suspension volume and homogenize with B. Braun homogenizer for 1 min × 3 times at 4˚C. |
Comment 0
|
|
5) |
Homogenates are filtrated by G1 glass filter and centrifuged at 10,000 × g for 20 min. |
Comment 0
|
|
6) |
Supernatant is further centrifuged at 100,000 × g for 1 h. |
Comment 0
|
|
7) |
Collect pellet as HSP (high speed pellet). |
Comment 0
|
|
8) |
HSP are resuspended in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol, and proteinase inhibitor mixture. |
Comment 0
|
|
9) |
A 200 μg protein of HSP is incubated in 50 mL of 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 0.6% Triton X-100, 0.5 mM 1-deoxy-mannojirimycin, 20 pmol Man8GlcNAc2-PA, 1 mM GDP-mannose at 30˚C for 30 min. |
Comment 0
|
|
10) |
Boil for 5 min and reaction mixture is centrifuged at 15,000 rpm for 5 min. |
Comment 0
|
|
11) |
Supernatant of reaction mixture is analyzed by HPLC. |
Comment 0
|
|
|
2. |
|
1) |
Equilibrium buffer is a mixture of 95 % of solvent A (200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 3 : 7) and 5% of Solvent B (200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 7 : 3). |
Comment 0
|
|
2) |
Elution conditions are as follows:
Column: Asahipak NH2P-50 (0.46 × 25 cm)
Column temperature: 25˚C
Flow rate: 1.0 mL/min
Elution condition: linear gradient of Solvent B from 5% to 45% between 0–50 min
Fluorescent detector: Excitation = 310 nm, Emission = 380 nm |
Comment 0
|
|
|
Figure & Legends |
Figure & Legends
Fig. 1. Analysis of Och1p activity. A; Man8GlcNAc2-PA (acceptor). B; Man9GlcNAc2-PA (Och1p reaction product). |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
Date of registration:2015-06-03 13:33:29 |
This work is licensed under Creative Commons Attribution-Non-Commercial Share Alike. Please include the following citation
How to Cite this Work in an article:
Nakayama, Ken-ichi,
(2015). GlycoPOD https://jcggdb.jp/GlycoPOD.
Web.4,5,2024 .
How to Cite this Work in Website:
Nakayama, Ken-ichi,
(2015).
Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p).
Retrieved 4,5,2024 ,
from https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t193.
html source
Nakayama, Ken-ichi,
(2015).
<b>Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p)</b>.
Retrieved 5 4,2024 ,
from <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t193" target="_blank">https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t193</a>.
Including references that appeared in the References tab in your work is
much appreciated.
For those who wish to reuse the figures/tables, please contact JCGGDB
management office (jcggdb-ml@aist.go.jp).
|
|