嗜热毛壳菌一种新的Cu,Zn superoxide dismutase的表达、纯化和结晶(英文)

【摘要】：正Chaetomium thermophilum cz1 gene was successfully over-expressed in P.pastoris GS115 when induced with methanol supplement.The expressed enzyme contains 154 amino acids with a calculated MW of 16.1 kDa based on the amino acid sequence.The recombinant CZ1 had the highest activity when induced after five days and the expression level was 2.56 mg ml-1.The expressed protein was purified from the culture filtrate with a single ion-exchange chromatography step.The purified enzyme showed an activity of about 127.4 U mg-l. Electrophoresis of the recombinant CZ1 on SDS-PAGE gave a single band with a molecular mass of about 17.7 kDa.This value is higher than 16.1 kDa estimated from the deduced amino acid sequence,suggesting that the recombinant CZ1 might be glycosylated.The availability of pure enzyme will allow a full biochemical and structural characterisation,including assessment of its thermal stability.The recombinant enzyme was crystallized at pH 8.2 using NaK phosphate 1.4 M as precipitant.Data to 1.9 A resolution were collected at 100 K in a synchrotron radiation source from a single crystal.The crystals belong to the P61/P65 space group with unit-cell dimensions a=90.2,b=90.2,c=314.5 A,and 8 molecules in the asymmetric unit.The presence of 8 molecules in the asymmetric unit may reveal new features in oligomer formation in SODs and provide insights into SOD assembly and stability in disease through comparative studies with the human enzyme.Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure-activity relationship,assembly and protein stability of CuZnSODs.