Purification of rhLY by cation-exchange chromatography and gel filtration chromatography.

<p>(A) Purification of rhLY by cation-exchange chromatography through the Hiscreen SP FF prepacked column. Lyophilized powder (20 g) from transgenic egg whites was dissolved in 20 mM sodium phosphate buffer (pH 8.5) and applied to the column. F, the flow through; P, the elution peak. (B) Examination of the flow-through and elution peak from the Hiscreen SP FF prepacked column by 15% SDS-PAGE and Coomassie blue. (C) Western blot of the flow-through and elution peak from the Hiscreen SP FF prepacked column. (D) Purification of rhLY from the first chromatographic step by the Mono S 5/50 GL prepacked column. The P fraction from the first step was loaded on the column after being desalted by 20 mM sodium phosphate buffer (pH 7.0). P1, P2, P3, P4 and Mix represented the elution peaks. (E) Examination of the elution peaks from the second chromatographic step by 15% SDS-PAGE and Coomassie blue staining. (F) Western blot of the elution peaks from the second step. (G) Gel-filtration chromatography of the partly purified rhLY from the second step through the Superdex^TM 75 10/300GL prepacked column. The P3 fraction from the second chromatographic step was applied to the column after being concentrated with an ultrafiltration centrifugal tube. P1 and P2 represented different elution peaks. (H) Examination of the rhLY from the third chromatographic step by 15% SDS-PAGE and Coomassie blue staining. (I) Western blot identification of the elution peaks from Gel-filtration chromatography. hLY, commercial hLY (positive control for anti-hLY); cLY, commercial cLY (positive control for anti-cLY).