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Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

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Title Statement Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase
 
Added Entry - Uncontrolled Name Bhuvanasundar, Renganathan
Ragavachetty, Nareshkumar Nagaraj
Singh, Naveen Kumar
Coral, Karunakaran
Deepa, Perinkulam Ravi
Sulochana, Konerirajapuram Natarajan
 
Uncontrolled Index Term Enzyme kinetics; Recombinant mLOX; Spectroscopy and structure stability
 
Summary, etc. Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies.
 
Publication, Distribution, Etc. Indian Journal of Biochemistry and Biophysics (IJBB)
2019-08-23 17:08:23
 
Electronic Location and Access application/pdf
http://op.niscair.res.in/index.php/IJBB/article/view/27656
 
Data Source Entry Indian Journal of Biochemistry and Biophysics (IJBB); ##issue.vol## 56, ##issue.no## 2 (2019): IJBB Vol.56(2) [April 2019]
 
Language Note en