Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin
| Autor: | Raja Sudhakaran, Nandini B Nataraj, Sunil Kumar Sukumaran, Ganesh Sambasivam |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Blood Glucose Protein Folding medicine.medical_treatment In silico Recombinant Fusion Proteins Peptide 01 natural sciences Biochemistry law.invention 03 medical and health sciences Thioredoxins Structural Biology law 010608 biotechnology medicine Escherichia coli Humans Amino Acid Sequence Cloning Molecular 030304 developmental biology Proinsulin chemistry.chemical_classification Inclusion Bodies 0303 health sciences Base Sequence Insulin General Medicine Fusion protein Amino acid Enteropeptidase chemistry Gene Expression Regulation Solubility Recombinant DNA Thioredoxin Chromatography Liquid |
| Zdroj: | Protein and peptide letters. 27(5) |
| ISSN: | 1875-5305 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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