Expression And Analysis Of Earthworm Fibrinolytic Enzyme (EFE) In Methylotrophic Yeast

It is a focus to study thrombosis, such as some thrombolytic enzymes. Earthworm Fibrinolytic Enzymes(EFE) are novel thrombolytic agents found in eartheworm during recent years. Belonging to serine proteases, the enzymes have a set of proteolytic enzymes present in the body of earthworm. They have long been used as antipyretic and diuretic purposes in traditional Chinese medicine. In recent years, we have separated 11-13 bands on PAGE from earthworm by affinity chromatography and amplified an entire coding sequence by PCR using earthworm cDNA as a template. The sequence was cloned and expressed in E. coli, but the expressed EFE was inclusion bodies.At the same time we subcloned the EFE gene into pPICQK vetor. The result plasmid, pPIC9K-EFE, was used to transform the host Pichia pastoris strain GS115. Methylotrophic yeast expression system has been one of the most successful foreign protein expression system until now. It does not have the endotoxin problem associated with bacteria and has the potential to perform many of the posttranslational modifications typically associated with higher eukaryotes.In this research, a lots of transformants were obtained by improved electroporation. To screen high-level expression of Pichia pastoris transformants by in situ two-film method, we purified the expressed protein from ?coli and immunized rabbits. As a result, we obtained many transformants expressing high-level EFE. We also electrotransporated the linear pPIC9K-EFE into HSA and yeast strain Pichia pastoris containing the integranted HSA gene and EFE gene was obtained by G418 screening. The expressed EFE was identified by western blotting. In order to enhance theexpression level of EFE in Pichiapastoris, we studied inducing conditions by orthogonal design in shake flasks including methanol concentration, pH and dissolved oxygen.