Wadzinski B E, Eisfelder B J, Peruski L F, Mumby M C, Johnson G L
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
J Biol Chem. 1992 Aug 25;267(24):16883-8.
Functional expression of recombinant wild-type phosphatase 2A catalytic subunit has been unsuccessful in the past. A nine-amino-acid peptide sequence (YP-YDVPDYA) derived from the influenza hemagglutinin protein was used to modify the NH2 and/or COOH terminus of the phosphatase 2A catalytic subunit. Addition of the nine-amino-acid sequence at the NH2 terminus allowed recombinant phosphatase 2A expression as a predominantly cytosolic phosphatase 2A enzyme. The 12CA5 monoclonal antibody that recognizes the nine-amino-acid hemagglutinin peptide sequence was used to immunoprecipitate the epitope-tagged phosphatase 2A catalytic subunit. Assay of the immunoprecipitated epitope-tagged phosphatase 2A demonstrated an okadaic acid-sensitive dephosphorylation of [32P] histone H1 and [32P]myelin basic protein similar to that measured with the wild-type enzyme. Functional phosphatase activity could be demonstrated for the NH2-terminal modified phosphatase 2A catalytic subunit following transient expression in COS cells or stable expression in Rat1a cells. In contrast, the COOH-terminal-modified phosphatase 2A catalytic subunit was very poorly expressed. The NH2-, COOH-modified subunit, having the nine-amino-acid hemagglutinin peptide sequence encoded at both termini of the polypeptide, was also expressed as a functional phosphatase 2A enzyme. Thus, NH2-terminal modification of the phosphatase 2A catalytic subunit results in a functional plasmid-expressed enzyme. The unique nine-amino-acid epitope-tag sequence also provides a method to easily resolve the recombinant phosphatase 2A from the endogenous wild-type gene product and related phosphatases expressed in cells.
过去，重组野生型磷酸酶2A催化亚基的功能表达一直未成功。源自流感血凝素蛋白的一个九氨基酸肽序列（YP - YDVPDYA）被用于修饰磷酸酶2A催化亚基的NH2和/或COOH末端。在NH2末端添加该九氨基酸序列使得重组磷酸酶2A作为主要定位于胞质的磷酸酶2A酶表达。识别该九氨基酸血凝素肽序列的12CA5单克隆抗体被用于免疫沉淀带有表位标签的磷酸酶2A催化亚基。对免疫沉淀的带有表位标签的磷酸酶2A的检测表明，[32P]组蛋白H1和[32P]髓鞘碱性蛋白的冈田酸敏感去磷酸化与野生型酶所测结果相似。在COS细胞中瞬时表达或在Rat1a细胞中稳定表达后，可证明NH2末端修饰的磷酸酶2A催化亚基具有功能性磷酸酶活性。相比之下，COOH末端修饰的磷酸酶2A催化亚基表达很差。在多肽两端都编码有九氨基酸血凝素肽序列的NH2 -、COOH修饰亚基也作为功能性磷酸酶2A酶表达。因此，磷酸酶2A催化亚基的NH2末端修饰产生了一种功能性的质粒表达酶。独特的九氨基酸表位标签序列还提供了一种方法，可轻松地将重组磷酸酶2A与内源性野生型基因产物以及细胞中表达的相关磷酸酶区分开来。
