The application of a novel thrombin sensitive site in genetic engineering

To obtain high efficiency of cleavage of thrombin in fusion protein containing alpha-ANP fused to Ref peptides, the linker sequence designed as VIAGR which was different from GVRGPR formerly used was studied. Plasmid pHL carrying the fusion gene Ref-NT-ANP downstream from PL promoter was derived from expression vector pLY1 by inserting the fragment of NT-ANP into it. The expression of fusion gene was induced at 42 degrees C, and the interested protein Ref-NT-ANP accumulated as inclusion bodies was isolated by gradient centrifuge and then dissolved in 7 mol/L guanidinehydrochloride (Gdn-HCl). After dilution, renaturation and dialyzation, the cleavage of thrombin was examined using samples with 1.1 mol/L Gdn-HC1 and samples free of Gdn-HCl respectively. Digestion result showed that the novel-adopted cleavage sequence was highly sensitive to thrombin when the substrate dissolved in 1.1 mol/L Gdn-HCl. The time needed for 87% cleavage (the ratio of substrate to thrombin was 50 micrograms/u) was less than 24 hours. This sequence described here which was specifically recognized by thrombin might be broadly applied in other fusion systems.