title: Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases.
abstract: Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.
authors.affiliation: Department of Gene Technologies, Pharma Preclinical Research, F. Hoffmann-La Roche AG, Basel, Switzerland. fiona.grueninger@roche.com
authors.forename: F
authors.initials: F
authors.lastname: Grueninger-Leitch
authors.forename: A
authors.initials: A
authors.lastname: D'Arcy
authors.forename: B
authors.initials: B
authors.lastname: D'Arcy
authors.forename: C
authors.initials: C
authors.lastname: Chène
mesh_terms: D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases