Hou W R, Hou Y L, Wu G F, Song Y, Su X L, Sun B, Li J
Key Laboratory of Southwest China Wildlife Resources Conservation, Ministry of Education, College of Life Science, China West Normal University, Nanchong.
Genet Mol Res. 2011;10(3):1576-88. doi: 10.4238/vol10-3gmr1159.
The ribosomal protein L9 (RPL9), a component of the large subunit of the ribosome, has an unusual structure, comprising two compact globular domains connected by an α-helix; it interacts with 23 S rRNA. To obtain information about rpL9 of Ailuropoda melanoleuca (the giant panda) we designed primers based on the known mammalian nucleotide sequence. RT-PCR and PCR strategies were employed to isolate cDNA and the rpL9 gene from A. melanoleuca; these were sequenced and analyzed. We overexpressed cDNA of the rpL9 gene in Escherichia coli BL21. The cloned cDNA fragment was 627 bp in length, containing an open reading frame of 579 bp. The deduced protein is composed of 192 amino acids, with an estimated molecular mass of 21.86 kDa and an isoelectric point of 10.36. The length of the genomic sequence is 3807 bp, including six exons and five introns. Based on alignment analysis, rpL9 has high similarity among species; we found 85% agreement of DNA and amino acid sequences with the other species that have been analyzed. Based on topology predictions, there are two N-glycosylation sites, five protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, two tyrosine kinase phosphorylation sites, three N-myristoylation sites, one amidation site, and one ribosomal protein L6 signature 2 in the L9 protein of A. melanoleuca. The rpL9 gene can be readily expressed in E. coli; it fuses with the N-terminal GST-tagged protein, giving rise to the accumulation of an expected 26.51-kDa polypeptide, which is in good agreement with the predicted molecular weight. This expression product could be used for purification and further study of its function.
核糖体蛋白L9（RPL9）是核糖体大亚基的一个组成部分，其结构不同寻常，由两个通过α螺旋连接的紧密球状结构域组成；它与23 S rRNA相互作用。为了获取大熊猫核糖体蛋白L9（rpL9）的相关信息，我们基于已知的哺乳动物核苷酸序列设计了引物。采用逆转录聚合酶链反应（RT-PCR）和聚合酶链反应（PCR）策略从大熊猫中分离cDNA和rpL9基因；对其进行测序和分析。我们在大肠杆菌BL21中过表达rpL9基因的cDNA。克隆的cDNA片段长度为627 bp，包含一个579 bp的开放阅读框。推导的蛋白质由192个氨基酸组成，估计分子量为21.86 kDa，等电点为10.36。基因组序列长度为3807 bp，包括六个外显子和五个内含子。基于比对分析，rpL9在物种间具有高度相似性；我们发现其DNA和氨基酸序列与其他已分析物种的一致性达85%。基于拓扑预测，大熊猫L9蛋白中有两个N-糖基化位点、五个蛋白激酶C磷酸化位点、一个酪蛋白激酶II磷酸化位点、两个酪氨酸激酶磷酸化位点、三个N-肉豆蔻酰化位点、一个酰胺化位点和一个核糖体蛋白L6特征2。rpL9基因能够在大肠杆菌中轻松表达；它与N端带有谷胱甘肽S-转移酶（GST）标签的蛋白融合，产生预期的26.51-kDa多肽积累，这与预测的分子量高度相符。该表达产物可用于纯化及其功能的进一步研究。
