Engineering and production of recombinant bovine pancreatic ribonuclease enzyme (RNase A) as a potential therapeutic

Document Type : Research Paper

Authors

1 Animal Science Department, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Professor, Recombinant proteins research group, The research institute of biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

3 Animal Science Department, Faculty of Agriculture, University of Guilan, Rasht, Iran

4 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

Objective 
Immunotoxins are one of the most promising ways in therapeutic fields, specially cancer therapy which have a unique toxin-antibody structure, and kill the cancer cell by passing through the cell membrane and entering the target cell. The aim of this study was to engineer and design bovine pancreatic enzyme (RNase A) for to escape from ribonuclease inhibitor (RI) as a toxin segment in designing of immunotoxins used in therapeutic fields.
 
 
 
Materials and Methods
After bioinformatics investigation using PyMol software, engineering and substitution of target amino acid in wild gene coding RNase A sequence (K91A) were performed. The wild type and recombinant proteins in E.coli (BL21(D3) strain) were expressed and refolded, and then approved using SDS-PAGE. The purification of produced proteins was conducted using an immobilized-metal affinity chromatography for His-tagged proteins.
 
Results
The results showed that the protein concentrations were 3 and 2.2 g/L for wild RNase A and recombinant enzyme. The hydrolysis activity measurement of the produced enzymes, in presence of different concentrations of RI, indicated that recombinant variant had lower sensitivity and higher catalytic activity in comparison with the wild enzyme. The results obtained from enzyme activity of recombinant enzyme compared to the wild enzyme, in presence or absence of RI, demonstrated that the interaction effect between RNase A and RI can be disrupted by manipulation of amino acids involved in this interaction.
 
Conclusion
In respect to the current results, it was concluded that RNase A enzyme can be used as a promising drug in engineering and production of immunotoxins.

Keywords

References

 
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Agricultural Biotechnology Journal
Volume 12, Issue 4
December 2020
Pages 60-76

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