Expression, Purification, And Activity Identification Of Snake Fibrinolytic Enzyme Alfimeprase In Pichia Pastoris

Nowadays thrombotic diseases are one of the most serious diseases that greatly affected people's life, such as peripheral arterial occlusion (PAO) and central venous access device occlusion (CVAD). Among all of the therapies, thrombolytic therapy is regarded as the most effective method to cure the above diseases. As reported, there are about 17 million thrombotic patients in the world every year and the potential thrombolytic need is about 2 billion dollars. Therefore, how to research and develop a native, effective thrombolytic and to achieve high level expression will play a significant and profound role in the antithrombotic processes.Alfimeprase (ALF) with the N-terminal sequence of SFPQR--, is a recombinantly modified variant of zinc metalloproteinase fibrolase, which was first isolated from the venom of the Southern copperhead snake (Agkistrodon contortrix contortrix). Like fibrolase, ALF directly and proteolyticly cleaves bothαandβchains of fibrin and fibrinogen and has no chain activity agains theγchains. Circulating ALF can be functionally inactivated by a2- macroglobulin in the form of rapid and irreversible covalent binding. Based on the biochemical characteristics and mechanism of ALF described above, investigations of PAO and CVAD occlusion were initiated by the Nuveo Company, which is the current national holder of the patent of ALF. The phase I trials to evaluate pharmacokinetics, thrombolytic activity in patients with chronic PAO and the phase II trials to evaluate the safety and efficiency profile in patients with CVAD occlusion have been accomplished, respectively. The results indicate that ALF is not only a direct-acting, dose-dependent and generally well tolerated thrombolytic, but also has the potential to be a novel, fast, safe and effective thrombolytic. Phase III studies in both acute PAO and CVAD occlusion are scheduled to begin in the near future.Since the Pichia pastoris (P. pastoris) expression system has the characteristic advantages of extremely high level expression of intra- or extracellular proteins, ease of genetic manipulation, ease of fermentation to high cell density and ease of the downstream purification, it is regarded as the most effective heterologous protein expression system. In the present report, the P. pastoris expression system was used to achieve a high level secretion and full activity expression of ALF. ALF coding sequence fused with a 6*?histidine tag and an enterokinase recognition site at the N-terminus was cloned into the expression vector of pPIC9K and pPICZαA and then highly transformed into the P. pastoris strains of GS115 and KM71 by electroporation. Selection, the genomic DNA PCR identification and methanol induction were then carried out to obtain the high level expression transformants of GS115/pPIC9K-his6-alf, KM71/pPIC9K-his6-alf and GS115/pPICZαA-his6-alf. SDS-PAGE and Western blotting analysis showed that the secreted recombinant ALF (rALF) had a molecular weight of 23.8 kDa and was bound specifically to the mouse anti-His·tag monoclonal antibody.This showed that rALF was specifically secreted in the above transformants. Optimization of the culture parameters of pH value, initial A600 value, methanol daily addition concentration and induction time length were also accomplished and under the optimized conditions, the production of rALF reached up to 510 mg/L, 465 mg/L and 425mg/L of the three above transformants, respectively. As to the pPIC9K-his6-alf transformants, it also appeared that KM71 was producing a more pure protein than GS115 while GS115 was producing more rALF per unit volume. Through one-step affinity chromatography, the purities of rALF from the three above transformants were as high as 94.8 %, 96 % and 95 %. The fibrinolytic activity of rALF revealed by the modified fibrin plate method demonstrated that the protein was efficiently secreted and functionally expressed, and thrombolysis of rALF was demonstrated to be dose-dependent and time-relative. Furthermore, analysis of the SDS-PAGE and mixed fibrinolytic activity identification results indicate that pPIC9K is more advantageous than pPICZαA as for the efficient and functional expression of rALF, while there is no such definite conclusion between the comparison of GS115 and KM71 strains.In conclusion, we have developed an efficient and functional expression system for rALF in this report, and the system may not only facilitate further studies of ALF and other thrombolytic agents, but also can allow possible large-scale production of ALF in the near future.