Title:Molecular Expression of Bioactive Recombinant Methionine Sulfoxide Reductase A (MsrA)
Volume: 28
Issue: 1
Author(s): M.S. Indhu, Shruthi Nanjundappa, Ramamoorthy Muttu, Upmanyu Vikramaditya, Manish Mahawar, Mihir Sarkar, Taru Sharma Guttula and Sanjeevkumar Bhure*
Affiliation:
- Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar - 243122, Bareilly, U.P.,India
Keywords:
Buffalo, recombinant MsrA, expression, enzyme activity, spectroscopic method, biochemical assay.
Abstract:
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.
