Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD2
Davis SJ, Puklavec MJ, Ashford DA, Harlos K, Jones EY, Stuart DI, Williams AF. (1993), Protein Eng. 6, 229-32
Key figure: Deglycosylation and crystallization of sCD2
(A) The soluble CD2 expressed in the Lec22.214.171.124 cell line was treated with endoglycosidase H and purified by Sephadex G-75 gel-filtration. The starting material (4 μg, lane 2) and deglycosylated sCD2 (4 μg, lane 3) were then analysed on a 15% SDS-Page gel run under reducing conditions and then stained with Coomassie blue. For comparison sCD2 expressed in CHO-K1 cells was also run (4 μg, lane 1). (B) The deglycosylated sCD2 was concentrated and subjected to hanging-drop, vapour-diffusion crystallization trials in the presence of 15-18% polyethylene glycol (Mr = 4000). Typical crystals are shown; the largest crystals grew to 0.3 × 0.08 × 0.08 mm.