重组人核苷二磷酸激酶A的理化性质

对重组人核苷二磷酸激酶A（rhNDPK-A）进行纯化，并对重组产物的理化性质及在溶液中的聚合状态进行鉴定。NDPK-A工程菌发酵后的菌体高压匀浆，然后微孔过滤、超滤浓缩，所得样品经DEAE阴离子交换、Cibacron Blue亲和层析、分子筛层析三步纯化后，以SDS-PAGE和RP-HPLC分析纯化产物的纯度，RP-HPLC测定酶活性。合格制品以基质辅助激光解析飞行时间质谱测定相对分子质量（MW）；Edman降解法测定N末端序列；多角度激光散射法测定重组产物在溶液中的表观分子量。结果表明，rhNDPK-A纯化产物的SDS-PAGE纯度为97.3%，RP-HPLC纯度为99.2%；比活性为（900±100）u/mg；单体相对分子质量为17017，与NDPKA分子量理论值相差132。测序结果表明，rhNDPK-A N末端缺失Met残基，其理论分子量为17017，与飞行质谱测定结果完全一致。表观分子量测定结果表明，rhNDPK-A在溶液中形成六聚体，表观分子量为102kD。上述结果说明， NDPK-A重组产物具与天然产物相同的自发形成六聚体性质，这为NDPK-A新药开发和机理研究打下了良好基础。

Physical and Chemical Characters of Recombinant Human Nucleoside Diphosphate Kinase A

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.