Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor.
Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor.
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储存颗粒蛋白23的纯化。
DOI：
10.1074/jbc.271.47.30126
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Schleef,RR
中科院分区：
文献类型：
--
作者：
Lang,IM;Chuang,TL;Barbas3rd,CF;Schleef,RR
1型纤溶酶原激活物抑制剂（派-1）是纤溶级联反应的关键调节剂，以快速释放的形式储存在血小板α颗粒内。为了鉴定可能参与靶向或储存这种有效抑制剂的蛋白质，本报告研究了利用丝状噬菌体展示由含有受调节的分泌途径的细胞表达的蛋白质及其基于与派-1相互作用的富集的适用性。为此，从AtT-20细胞中提取RNA（即用于细胞内蛋白质分选的经典模型细胞系统），逆转录，使用含有内部限制性位点的聚合酶链反应引物扩增，并克隆到噬菌粒pCOMB 3 H中以作为与噬菌体基因III蛋白的融合构建体表达。并通过针对固相和液相派-1淘选来富集所得AtT-20 cDNA-噬菌体文库。将富集的cDNA文库亚克隆到原核表达载体系统中，所述原核表达载体系统用十肽标签替换基因III蛋白用于免疫定量。将一个新的cDNA克隆（即A-61）亚克隆到原核表达载体pTrcHis中，构建了一个含有N-末端6-组氨酸纯化标签的构建体，该cDNA克隆优先识别溶液相派-1，并与α-颗粒免疫兔产生的抗体反应阳性。该构建体在E.大肠杆菌，通过镍螯合层析纯化，然后进行制备性SDS-聚丙烯酰胺凝胶电泳，并用于产生多克隆抗体。免疫印迹分析采用抗体对纯化的A-61构建体揭示了一个23 kDa的蛋白存在于调节分泌途径的AtT-20细胞。经DEAE-Sephacel离子交换层析、Sephacryl S-100分子筛层析、Polybuffer exchanger 94聚焦层析和派-1-Sepharose亲和层析，从AtT-20细胞条件培养液中纯化出23-kDa分子。采用23-kDa贮藏颗粒蛋白的16-kDa Lys-C蛋白水解片段的N-末端氨基酸测序来确认其与克隆A-61的cDNA序列的同一性。这些数据表明，融合到基因III蛋白的C-末端区域的cDNA文库的噬菌体展示及其通过与靶分子相互作用的富集可用于确定特定细胞途径内存在的其他蛋白。
Type 1 plasminogen activator inhibitor (PAI-1) is a key regulator of the fibrinolytic cascade that is stored in a rapidly releasable form within platelet α-granules. To identify proteins that may participate in the targeting or storage of this potent inhibitor, this report investigates the applicability of utilizing filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and their enrichment based upon an interaction with PAI-1. For this purpose, RNA was extracted from AtT-20 cells (i.e.a classical model cell system for intracellular protein sorting), reverse transcribed, amplified using polymerase chain reaction primers containing internal restriction sites, and cloned into the phagemid pCOMB3H for expression as fusion constructs with the bacteriophage gene III protein.Escherichia coliwas transformed with the phagemids and infected with VCSM13 helper phage, and the resulting AtT-20 cDNA-bacteriophage library was enriched by panning against solid- and solution-phase PAI-1. The enriched cDNA library was subcloned into a prokaryotic expression vector system that replaces the gene III protein with a decapeptide tag for immunologic quantitation. One novel cDNA clone (i.e.A-61), which preferentially recognized solution-phase PAI-1 and reacted positively with antibodies derived from a rabbit immunized with α-granules, was subcloned into the prokaryotic expression vector pTrcHis to create a construct containing an N-terminal six-histidine purification tag. This construct was expressed inE. coli, purified by nickel-chelate chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and utilized for the generation of polyclonal antibodies. Immunoblotting analysis employing antibodies against the purified A-61 construct revealed a 23-kDa protein present in the regulated secretory pathway of AtT-20 cells. The 23-kDa molecule was purified from media conditioned by AtT-20 cells by ion exchange chromatography on DEAE-Sephacel, molecular sieve chromatography on Sephacryl S-100, chromatofocusing on Polybuffer exchanger 94, and affinity chromatography on PAI-1-Sepharose. N-terminal amino acid sequencing of a 16-kDa Lys-C proteolytic fragment of the 23-kDa storage granule protein was employed to confirm its identity with the cDNA sequence of clone A-61. These data indicate that phage display of cDNA libraries fused to the C-terminal region of the gene III protein and their enrichment via an interaction with a target molecule can be utilized to define other proteins present within a particular cellular pathway.