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Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin.
Protein & Peptide Letters ( IF 1.6 ) Pub Date : 2020-05-01 , DOI: 10.2174/0929866526666191028150843
Nandini B Nataraj 1, 2 , Sunil Kumar Sukumaran 1, 2 , Ganesh Sambasivam 1, 2 , Raja Sudhakaran 2
Affiliation  

Background: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process.

Objective: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield.

Methods: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification.

Results: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 – 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps.

Conclusion: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.



中文翻译:

截短的硫氧还蛋白肽可作为胰岛素原生产的高效融合标签。

背景:胰岛素是一种用于调节血糖水平的肽激素。预计人类胰岛素市场将以每年12.5%的速度增长。为了满足患者的需求,必须开发一种具有成本效益的胰岛素生产策略。这可以通过选择合适的宿主,理想的融合标签和简化的下游过程来实现。

目的:在本文中,我们证明了选择合适的融合伴侣来表达有毒蛋白(如胰岛素)在提高重组蛋白产量中起着重要作用。

方法:在本文中,我们重点研究了通过截短硫氧还蛋白标签来鉴定表达胰岛素原的肽标签融合伴侣。从蛋白质的氨基末端和羧基末端都进行了截短,并通过用胰岛素原基因表达截短序列来评估截短序列的效率。融合至胰岛素原的FCTRX(1-15)序列被进一步加工以建立下游纯化方案。

结果:考虑到融合标签:蛋白质比例,硫氧还蛋白标签被适当地截短。根据其计算机特性,鉴定了几个10到15个氨基酸的序列。这些FCRTX(1-15)显示融合蛋白的表达和稳定性增加。经酶促转化和色谱分离步骤后,从1g包涵体中生成156 mg纯化的胰岛素。

结论:根据目前的研究结果,得出的结论是FCTRX(1-15)肽具有被认为是表达胰岛素原的理想融合标签的有利属性。通过与其他蛋白质一起表达,可以进一步探索。

更新日期:2020-05-01
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