毕赤酵母表达重组hPK-5蛋白不均一性的鉴定  

Characterization of Heterogeneity of Recombinant Human Plasminogen Kringle 5 Expressed by Pichia pastoris

作　　者：李永红[1] 饶春明[1] 史新昌[1] 陶磊[1] 袁力勇[1] 王军志[1] 

机构地区：[1]中国药品生物制品检定所,北京100050

出　　处：《药物生物技术》2008年第3期180-184,共5页Pharmaceutical Biotechnology

基　　金：国家高技术研究发展计划(863计划)资助项目(2003AA2Z3480)

摘　　要：对毕赤酵母表达重组人纤溶酶原Kringle 5（hPK-5）蛋白的不均一性进行了鉴定。应用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）法及分子排阻高效液相色谱（SEC-HPLC）和反相高效液相色谱（RP-HPLC）进行纯度分析。采用糖蛋白染色法鉴定是否为糖蛋白，以Edman降解法测定N端氨基酸，采用高效液相色谱-电喷雾四极杆飞行时间质谱（HPLC-ESI-QTOF-MS）联用技术测定蛋白质精确分子质量。结果表明：SDS-PAGE测定为单-条带，纯度大于95％，SEC-HPLC测定为单一色谱峰，纯度大于95％，RP-HPLC测定为几个紧密相连的峰。糖蛋白染色为阴性。N端氨基酸测定结果表明含多个N端氨基酸。液质联用测定精确分子质量的结果表明hPK-5蛋白是相对分子质量分别为10744．88，10646．00，10533．00和10419．63的混合物。以上结果准确鉴定出毕赤酵母表达的该重组hPK-5蛋白是C端完整而N端依次缺失0～3个氨基酸的不均一蛋白。The heterogeneity of recombinant human plasminogen kringle 5 （hRK-5） expressed secretorily in Pichia pastoris was investigated. The purity was analyzed by sodium dodeeyl sulfate polyaerylamide gel electro-potation （SDS-PAGE）, size-exclusion high performance liquid chromatography （SEC-HPLC） and reversed-phase high performance liquid chromatography （RP-HPLC）. Glycoprotein staining was used for glyeoprotein characterization. N terminal amino acid sequence was determined by the method of Edman degradation. Precise molecular mass of hPK-5 was detremined by high-performance liquid chromatography coupled with eleetrospray ionization quadrupole time-of-flight mass spectrometry （HPLC-ESI-Q-TOF-MS）. Both purity results detected by SDS-PAGE and SEC-HPLC were more than 95%. However RP-HPLC chromatogram showed that there were several connective peaks, which indicated that the recombinant hPK-5 protein may be a mixture of different forms. Glyeoprotein staining showed that recombinant hPK-5 protein was not glycoprotein. N terminal amino acid sequencing indicated that there were several N terminus amino acids. Next, recombinant hPK-5 was characterized by HPLG-ESI-Q-TOF-MS and found that hPK-5 was actually a mixture of four polypeptides whose relative moleeaular weights were 10 744.88,10 646.00,10 533. 00 and 10 419.63 respectively. All the results indicated that the recombinant hPK-5 protein expressed by Pfchia pastoris was a heterologous mixture and respectively lacked 0-3 N-terminal amino acids.

关 键 词：hPK-5 毕赤酵母 重组蛋白 不均一性 

分 类 号：R917[医药卫生—药物分析学]