Title
Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus
Author
Dhaval K Patel, Kunal R Shah, Anju Pappachan, Sarita Gupta, Desh Deepak Singh
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis. It is also referred to as a moonlighting protein as it has many diverse functions like regulation of apoptosis, iron homeostasis, cell-matrix interactions, adherence to human colon etc. apart from its principal role in glycolysis. Lactobacilli are lactic acid bacteria which colonize the human gut and confer various health benefits to humans. In the present study, we have cloned, expressed and purified the GAPDH fromLactobacillus acidophilus to get a recombinant product (r-LaGAPDH) and characterized it. Size exclusion chromatography shows that r-LaGAPDH exists as a tetramer in solution and have a mucin binding and hemagglutination activity indicating carbohydrate like binding adhesion mechanism. Fluorescence spectroscopy studies showed an interaction of r-LaGAPDH with mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine with a Kd of 3.6 ± 0.7 × 10−3 M, 4.34 ± 0.09 × 10−3 M, 4 ± 0.87 × 10−3M and 3.7 ± 0.28 × 10−3 M respectively. We hope that this preliminary data will generate more interest in further elucidation of the roles of GAPDH in the adhesion processes of the bacteria.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Thermal stability, Protein folding, Biochemistry