水蛭素12肽与瑞替普酶融合蛋白的反相色谱纯化及其一级结构确认

RP-HPLC Purification for Primary Structure Confirmation of 12 Peptides of Hirudin and Reteplase Fusion Protein

目的探索水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的高效液相色谱分离纯化方法,确认其一级结构。方法比较PST、Source 30、PLHP 3种不同的反相色谱介质纯化HV12p-rPA的性能,并建立反相分离纯化方法。用MALDI-TOF-MS法测定HV12p-rPA的相对分子质量(Mr),采用高效液相色谱-质谱联用法(HPLC-MS),分离与分析该蛋白质经胰蛋白酶、胰凝乳蛋白酶酶切后得到的多肽混合物,通过二级质谱和数据库检索对分离出的各个肽段进行序列鉴定。结果PST色谱介质性能最好。在流速2.0mL/min条件下,采用线性梯度洗脱得到的目标蛋白质样品纯度≥95%。测得该蛋白质的精确Mr为41473,与其理论Mr相符。肽段的实验检测值与理论平均值的数据比对表明,已检出肽段的总覆盖率≥85%。结论确定了HV12p-rPA蛋白质的反相分离纯化方法,验证了该蛋白质一级结构正确。

Objective To explore the method for the separation and purification of 12 peptides of hirudin and reteplase fusion protein（HV12p-rPA） by RP-HPLC and determine its primary structure. Methods Three kinds of high performance reversed phase chromatographic matrix, namely PST, Source 30 and PLHP were employed and different conditions were tried to purify the fusion protein for the confirmation of its primary structure. The relative molecular mass （Mr） of the eluted fusion protein was measured by MODI-TOF-MS. The fusion protein was then digested with trypsin and chymotrypsin, and the peptide fragments were analyzed by HPLC-MS. The amino acid sequence of peptide fragments was determined by the combination of secondary mass spectra and database retrieval. Results The PST column was more highly resolved and a relatively better condition was achieved by increasing mobile phase B （0.1% trifluoroacetic acid ＋ acetonitrile） from 20% to 80% in 40min at a flow rate of 2.0mL/min. The purity of the eluted fusion protein was up to 95%, and its M measured by MODI-TOF was 41 473, which was in accordance with the theoretical value. 85% of the expected peptides were covered by the protein database. Conclusion The RP-HPLC for the separation and purification of HV12p-rPA can be established and its primary structure can be validated.