On searching the target proteins for mastoparan from bovine brain

在本研究中，我们计划作为一个亲和配体的mastoparan类似物（LLL-mastoparan-G），其中mastoparan的N-末端区域的修饰和C-末端区域的Gly延伸。使用肽合成仪通过逐步延伸法制备具有配体的亲和树脂，其方式是从C-末端到N-末端氨基酸顺序地将其偶联到氨基树脂。裂解后得到的亲和配体的结构通过使用肽测序仪和高分辨率正FAB-MS光谱仪进行分析。亲和层析和SDS-PAGE结果表明，钙调素在钙离子存在下能与亲和配体结合。此外，在开柱型亲和层析的基础上，设计了高效液相色谱型亲和层析，利用新制备的亲和树脂对牛脑中的乳癖菌素进行了目标蛋白的筛选。我们的亲和层析系统表明，独特的蛋白质结合的亲和配体只有在钙离子的存在下。用Max-yield电泳仪洗脱蛋白质条带，或用Western印迹法将蛋白质条带转移到PVDF膜上。还尝试了以上述方式洗脱的蛋白质的氨基酸分析和蛋白质序列分析。

In this study, we planned mastoparan analogue(LLL-mastoparan-G) as an affinity ligand in which the N-terminal region of mastoparan is modified and the C-terminal region is extended by Gly. The affinity resin with the ligand was prepared by stepwise elongation method using the peptide synthesizer, in a manner that it is coupled to amino resin sequentially from the C-terminal to the N-terminal amino acid. The structure of the affinity ligand obtained after cleavage was analyzed by use of peptide sequencer and high-resolution positive FAB-MS spectrometer. The data for peptide sequence and elemental composition corresponded closely to theoretical ones.It was shown by affinity chromatography and SDS-PAGE that calmodulin bound to the affinity ligand in the presence of calcium ion. Furthermore, HPLC-type affinity chromatography was devised in addition to the open column type.We searched for the target proteins for mastoparan from bovine brain by newly prepared affinity resin. Our affinity chromatography system showed that unique proteins bound to the affinity-ligand only in the presence of calcium ion. Its molecular weight was estimated as about 26k Da. The detected proteins bands were eluted from gels by use of Max-yield electroeluter, or transferred to PVDF membrane by western blotting. Amino acid analysis and protein sequence analysis of the proteins eluted in the manner described above was also attempted.