X-ray crystal structure determination of mammalian glycosyltransferases.
The vast majority of mammalian glycosyltransferases are endoplasmic reticulum (ER) and Golgi resident type II membrane proteins. As such, producing large quantities of properly folded and active enzymes for X-ray crystallographic analysis is a challenge. Described here are the methods that we have developed to facilitate the structural characterization of these enzymes. The approach involves the production of a soluble Protein A-tagged form of the catalytic domain in a mammalian cell expression system. Production is scaled up in a perfusion-fed bioreactor with media flow rates of 3-5 liters/day. Expression levels are typically in the 1- to 4-mg/liter range and a simple and efficient purification method based on immunoglobulin G (IgG)-Sepharose affinity chromatography has been developed. Our approach to delimiting the catalytic domain and deglycosylating it when necessary is also discussed. Finally, we describe the selenomethionine labeling protocol used in our X-ray crystal structure determination of leukocyte-type Core 2 beta1,6-N-acetylglucosaminyltransferase.
Pak
John E
JE
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Rini
James M
JM
D000818:Animals; D019149:Bioreactors; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D018360:Crystallography, X-Ray; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D012645:Selenomethionine / Q000737:chemistry; D016415:Sequence Alignment; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics
D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D012645:Selenomethionine; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase