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. Author manuscript; available in PMC: 2008 Dec 8.
Published in final edited form as: Mol Ther. 2007 Jul 24;15(10):1856–1862. doi: 10.1038/sj.mt.6300268

Figure 3. Intein-mediated factor VIII (FVIII) light chain ligation.

Figure 3

(a) An illustration of the intein strategy. The top box with the dotted line shows the DNA manipulation procedures. The bottom box with the solid line shows the protein ligation reactions. (b) Western blot of intein-mediated ligation of the heavy chain (HC) and light chain (LC). Plasmids pCMV-BDD-FVIII, pCMV-HC-INT, pCMV-LC-INT, and pCMV-HC-INT+pCMV-LC-INT at ratios of 9:1, 1:1, and 1:9 were transfected into COS cells using Lipofectamine. At 36 hours after transfection, cell lysates were prepared for Western blot analysis (4–12% gradient polyacrylamide gel electrophoresis gel). The blot was probed with a sheep anti-human FVIII polyclonal antibody (Haematologic Technologies, VT). Lanes are: 1: ReFacto FVIII protein; 2: mock transfection; 3: pCMV-BDD-FVIII; 4: pCMV-LC-INT; 5: pCMV-HC-INT; 6: pCMV-LC-INT+pCMV-HC-INT at a ratio of 9:1; 7: pCMV-LC-INT+pCMV-HC-INT at 1:1; 8: pCMV-LC-INT+pCMV-HC-INT at 1:9. cDNA, complementary DNA; SC: single-chain B-domain deleted (BDD)-FVIII protein.