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Molecular expression of bioactive recombinant methionine sulfoxide reductase A (MsrA)

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Title Molecular expression of bioactive recombinant methionine sulfoxide reductase A (MsrA)
Not Available
 
Creator M.S. Indhu
Shruthi Nanjundappa
Ramamoorthy Muttue
Upmanyu Vikramaditya
Manish Mahawar
Mihir Sarkar
Taru Sharma Guttula
Sanjeevkumar Bhure
 
Subject Buffalo
biochemical assay
enzyme activity
expression
recombinant MsrA
spectroscopic method
 
Description Not Available
Background: The increase in reactive oxygen species (ROS) production during cryopreservation of semen, leads to oxidation of biomolecules affecting the functionality of spermatozoa. Methionine residues in proteins are highly prone to oxidation and get converted into methionine sulfoxide (MetO). Methionine sulfoxide reductase A (MsrA) can improve the functionality of spermatozoa by reducing the MetO to methionine restoring the lost functionality of the affected proteins.

Objective: The expression of catalytically active recombinant MsrA (rMsrA).

Methods: The msrA gene was PCR amplified, cloned and sequenced. Further, the recombinant clone was used for protein expression and purification. The protein was getting precipitated during dialysis in Tris-buffer. Hence, the purified rMsrA was dialyzed at 4°C against the Tris-buffer pH 7.5 containing MgCl2, KCl, NaCl, urea and triton X-100. During dialysis, changes of buffer were done at every 12 h interval with stepwise reduction in the concentrations of NaCl, urea and triton X-100. The final dialysis was done with buffer containing 10 mM MgCl2, 30 mM KCl, and 150 mM NaCl, 25 mM Tris-HCl pH 7.5. The activity of the rMsrA was checked spectrophotometrically.

Results: The protein BLAST of buffalo MsrA with bovine sequence showed 14 amino acid mismatches. The rMsrA has been purified under denaturing conditions as it was forming inclusion bodies consistently during protein expression. After renaturation, the purified 33 kDa rMsrA was catalytically active by biochemical assay.

Conclusion: The rMsrA expressed in prokaryotic system is catalytically active and can be used for supplementation to semen extender to repair the oxidatively damaged seminal plasma proteins that occur during cryopreservation.
Not Available
 
Date 2023-05-26T03:28:31Z
2023-05-26T03:28:31Z
2020-05-08
 
Type Research Paper
 
Identifier Not Available
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/77757
 
Language English
 
Relation Not Available;
 
Publisher BenthamScience