Liao Ming Juan, Zhu Mu Yuan, Zheng Xu, Zhang Zhi He, Zhang An Ju
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310012 PR China.
Comp Biochem Physiol B Biochem Mol Biol. 2003 May;135(1):109-16.
A cDNA encoding Ailuropoda melanoleuca growth hormone (AmGH) was isolated from pituitary total RNA using RT-PCR and expressed in Escherichia coli. This is the first report of a GH nucleotide and amino acid (aa) sequence from giant panda. The open reading frame of AmGH (651 bp) encodes a precursor of 216 aa comprising a 26 aa signal peptide and a 190 aa mature protein with four cysteine residues similar to the typical primary structure of mammalian GH precursor. AmGH shares a high degree of identity (54-98.9%) with that of mammals, birds and amphibians, but a very low identity with bony fish GH (only 20-30%). The mature AmGH exhibits striking similarity to that of putative ancestral GH with a difference of only two residues, indicating a very slow basal rate of molecular evolution. The DNA fragment encoding mature AmGH was then subcloned into the pGEX-4T-1 expression vector and highly expressed in E. coli host BL21 with IPTG induction. The expressed proteins fused to GST were found to be sequestered into inclusion bodies and therefore the NaOH method was employed to solubilize the inclusion bodies; the proteins were further purified by Glutathione Sepharose 4B affinity chromatography. The production and purification of GST-AmGH reported here provide a basis for further studies on the biological activity of AmGH.
利用逆转录聚合酶链反应（RT-PCR）从大熊猫垂体总RNA中分离出编码大熊猫生长激素（AmGH）的互补DNA（cDNA），并在大肠杆菌中进行表达。这是关于大熊猫生长激素核苷酸和氨基酸序列的首次报道。AmGH的开放阅读框（651bp）编码一个由216个氨基酸组成的前体，其中包括一个26个氨基酸的信号肽和一个190个氨基酸的成熟蛋白，该成熟蛋白含有四个半胱氨酸残基，类似于哺乳动物生长激素前体的典型一级结构。AmGH与哺乳动物、鸟类和两栖动物的生长激素具有高度的同源性（54%-98.9%），但与硬骨鱼生长激素的同源性非常低（仅20%-30%）。成熟的AmGH与推测的祖先生长激素具有惊人的相似性，仅相差两个残基，这表明其分子进化的基础速率非常缓慢。然后将编码成熟AmGH的DNA片段亚克隆到pGEX-4T-1表达载体中，并在大肠杆菌宿主BL21中通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导进行高效表达。发现与谷胱甘肽S-转移酶（GST）融合表达的蛋白被隔离在包涵体中，因此采用氢氧化钠法溶解包涵体；蛋白再通过谷胱甘肽琼脂糖4B亲和层析进一步纯化。本文报道的GST-AmGH的制备和纯化方法为进一步研究AmGH的生物学活性奠定了基础。
