Novel purification method based on Fe3O4@SiO2@Ni (OH)2 magnetic nanoparticle and NpuDnaE Intein for high pure Streptokinase purification

Background and Objective: Streptokinase (SK) is the leading medicine for acute myocardial infarction treatment in developing countries. Industrial production of SK is a multi-step, high cost, onerous and time-consuming. Affinity tags are appropriate for highly pure protein purification but they need affinity column and should be removed from the final product by special proteases purification method. Inteins are the fast, precise and low-cost alternative for proteases-based tag removal. Nostoc punctiforme DnaE intein is a well-studied intein which has been modified in many demanding ways. Magnetic nanoparticles can separate tagged protein directly from the cell lysate rapidly and dispel the need for affinity column. Here we described a novel purification method for high pure and tagless Streptokinase (SK) production using Fe3O2@SioNi (OH) 2magnetic nanoparticle and NpuDnaE intein. Materials and Methods: In the present study, NpuC-SK and NpuN-His cloned in pET28 and pET21. Then expressed in E.coli BL21 (DE3). Fe3O2@SioNi (OH) 2magnetic nanoparticle synthesized by Co-precipitation and Solvothermal method and analyzed by XRD, FTIR, and VSM for size, structure and magnetization ability respectively. NpuN-His protein separated by Fe3O2@SioNi (OH) 2using neodymium external magnetic force directly from the cell lysate. After washes, expressed NpuC-SK cell lysate loaded on pre-captured NpuN-His by Fe3O2@SioNi (OH) 2. After 15min NpuC-SK attached to NpuN-His, washed then 5mM DTT (dithiothreitol) administrated to triggered SK cleaving and elution. Purified SK analyzed by SDS-PAGE and western blot then biological activity demonstrated by Chromogenic assay by S-2251 (H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride) substrate and Streptase® as standard. Findings: XRD and FTIR analyze suggested that Fe3O4@SiO2@Ni (OH) 2 structure with the size of 150-250 nm, also VSM showed that specific magnetization saturation value was 3.89 emu/g. Our result demonstrated that Fe3O2@SioNi (OH) 2capacity for capturing for NpuN-His was per nanoparticle. Up to 240 mg of nanoparticle could be dissolved in 1ml buffer. The highest spliced SK achieved after 3h incubation with DTT was up to ~88% but after 1h incubation spliced SK was accomplished to an acceptable yield up to ~84% SK inclusion bodies purification by NpuN/nanoparticles system yield 0/137±0.009mg/mg SK per NpuN*/nanoparticles. SDS-PAGE analyze of purified SK indicated that the purity of intein/nanoparticles-based purification was ~94%. Western blotting analyzes of the purified SK represented that the particular unspecific bond caused ~6% impurity was NpuN*-His fragment. Purified SK with this method was 88% biologically active comparing to Streptase.® Conclusion: The combination of NpuDnaE intein and magnetic nanoparticle resulted in the elimination of nonspecific protein attachment to the Ni2+ then reducing the final impurities, removing the His tag from the target protein, enhancing the final yield finally reducing the cost and time.

کلیدواژه ها:

Intein ، Magnetic nanoparticle ، Streptokinase (SK) ، acute myocardial infarction ، protein purification