Bakhtiari Nahid, Amini Bayat Zahra, Sagharidouz Sepideh, Vaez Mohsen
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran.
Avicenna J Med Biotechnol. 2017 Jan-Mar;9(1):19-22.
Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone (1-34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hPTH.
Novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a His-tag in N-terminal, 53 amino acids belong to β-galactosidase (LacZ) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an Enteropeptidase cleavage site, rhPTH (1-34) gene fragment. Optimized fusion gene was synthesized and ligated into pET-28a vector under control of T7 promoter, and then transformed in (DH5α) cells. Positive clones containing this gene were double digested with NcoI and-BamHI and also approved by sequencing. Gene overexpression was observed in SDS-PAGE after induction with 0.2 IPTG. Confirmation of gene expression was performed by western blotting using anti-His-tag antibody conjugated with peroxidase.
By this fusion gene design approach, we achieved a high level expression of the rhPTH, where it represented at least 43.7% of the total protein as determined by SDS-PAGE and confirmed by western blotting.
In addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial β-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhPTH with relevant isoelectronic point (pI). This innovation resulted in recombinant production of hPTH very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue.
甲状旁腺激素是由甲状旁腺分泌的一种含84个氨基酸的肽。其生理作用是维持正常的血清钙水平和骨重塑。该激素的生物活性与N端的1 - 34个氨基酸有关。激素（1 - 34）的重组形式自2002年起被批准用于治疗骨质疏松症。在本研究中，开发了一种新型融合伴侣，用于制备高产率的重组人甲状旁腺激素1 - 34氨基酸。
设计了一种新型核苷酸盒，编码一种嵌合融合蛋白，该融合蛋白包含一个N端带有His标签的融合伴侣、属于β - 半乳糖苷酶（LacZ）基因的53个氨基酸、一个用于提高表达和保护目标肽结构免受融合标签影响的接头序列、一个肠肽酶切割位点、重组人甲状旁腺激素（1 - 34）基因片段。合成优化后的融合基因，并将其连接到T7启动子控制下的pET - 28a载体中，然后转化到（DH5α）细胞中。用NcoI和 - BamHI对含有该基因的阳性克隆进行双酶切，并通过测序进行验证。用0.2 mM IPTG诱导后，在SDS - PAGE中观察到基因的过表达。使用与过氧化物酶偶联的抗His标签抗体通过蛋白质印迹法进行基因表达的确认。
通过这种融合基因设计方法，我们实现了重组人甲状旁腺激素的高水平表达，通过SDS - PAGE测定并经蛋白质印迹法确认，其占总蛋白的比例至少为43.7%。
除了在本研究中设计基因的高水平表达外，选择细菌β - 半乳糖苷酶的特定氨基酸序列作为载体标签的主要部分，用于保护该激素，这是具有相关等电点（pI）的重组人甲状旁腺激素的重要步骤。这一创新使得重组人甲状旁腺激素的生产效果良好，该基因构建体可作为类似重组肽的模板，其中重组蛋白降解是一个关键问题。
