FORMATION OF NUCLEOPHOSMIN B23 OLIGOMERS REQUIRES BOTH THE AMINO-TERMINAL AND THE CARBOXYL-TERMINAL DOMAINS OF THE PROTEIN
FORMATION OF NUCLEOPHOSMIN B23 OLIGOMERS REQUIRES BOTH THE AMINO-TERMINAL AND THE CARBOXYL-TERMINAL DOMAINS OF THE PROTEIN
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DOI：
10.1111/j.1432-1033.1991.tb16236.x
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发表时间：
1991-09-15
期刊：
影响因子：
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通讯作者：
CHAN, PK
中科院分区：
文献类型：
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作者：
LIU, QR;CHAN, PK
核磷蛋白/B23 是一种核仁磷蛋白，可形成寡聚体。 为了确定寡聚体形成所必需的结构域，构建了核磷蛋白/B23的各种缺失和点突变克隆。 核磷蛋白/B23 和突变蛋白是通过 (a) 耦合体外转录和翻译以及 (b) 使用 T7 RNA 聚合酶表达载体 (pET-8c) 在大肠杆菌中表达来产生的。 体外合成的核磷蛋白/B23 具有与 HeLa 细胞中合成的相同的肽图。 类似地，它形成可在 SDS/PAGE 中检测到的寡聚物，并在体内与氮芥交联。 用 Leu 取代 Met5、Met7 和 Met9 或删除 C 末端的 5 个氨基酸可消除寡聚化。 分子中部氨基酸部分（氨基酸残基83-152、117-186和185-240）的缺失对寡聚化影响很小。 N端和C端突变体克隆在体外的共表达没有产生寡聚物。 这些结果表明，N 端和 C 端结构域的分子内相互作用对于寡聚物的形成至关重要。
Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET-8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross-linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C-terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83-152, 117-186 and 185-240) had little effect on the oligomerization. Co-expression of the N- and C-terminal mutant clones in vitro did not produce oligomers. These results indicate that intra-molecular interactions with both the N- and C-terminal domains are essential for oligomer formation.