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. 2005 Apr 1;61(Pt 4):435-8.
doi: 10.1107/S1744309105008869. Epub 2005 Apr 1.

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

F J López-Jaramillo  1 , F Pérez-Banderas, F Hernández-Mateo, F Santoyo-González
Affiliations

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

F J López-Jaramillo et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.

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Figures

Figure 1
Figure 1
Synthesis of glucopyranosyl vinylsulfone from 1,2,3,4,6-penta-O-acetyl-β-d-glucopyranose. Reagents and conditions are as follows. (i) Thiourea, BF3Et2O, acetonitrile, reflux for 3 h then BrCH2CH2Cl, Et3N, room temperature, 2 h. (ii) Et3N, anhydrous MeOH, 313 K, 8 h. (iii) H2O2, AcOH, room temperature, 2 d. (iv) K2CO3, acetone, reflux, 8 h.
Figure 2
Figure 2
Native electrophoresis in 12% polyacrylamide gel. From left to right the samples are for the reaction after 24, 48 and 72 h, and for control HEW lysozyme.
Figure 3
Figure 3
Clusters of needle crystals (a) and the fragment used for diffraction (b) in a 0.3–0.4 mm loop.
Figure 4
Figure 4
2mF o − DF c map contoured at 1.2σ (blue) and 4σ (red) showing the electron density that defines the sugar bonded to Lys96 (a) and His15 (b).
See this image and copyright information in PMC

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