Molecular cloning, overexpression and characterization of human interleukin 1 alpha

by Rajalingam, Dakshinamurthy; Kacer, Doreen; Prudovsky, Igor; Kumar, Thallapuranam Krishnaswamy Suresh

Interleukin-1 alpha (IL-1 alpha) regulates a wide range of important cellular processes. In this study for the first time, we report the cloning, expression, biophysical, and biological characterization of the human interleukin-l alpha. Human IL-1 alpha has been expressed in Escherichia coli in high yields (similar to 4 mg per liter of the bacterial culture). The protein was purified to homogeneity (similar to 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1 alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1 alpha a is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1 alpha binds strongly (K-d similar to 5.6 x 10(-7) M) to S100A 13, a calcium binding protein that chaperones the in vivo release of IL-1 alpha into the extracellular compartment. Recombinant IL-1 alpha was observed to exhibit strong cytostatic effect on human umbilical vascular endothelial cells. The findings of the present study not only pave way for an in-depth structural investigation of the molecular mechanism(s) underlying the non-classical release of IL-1 alpha but also provide avenues for the rational design of potent inhibitors against IL-1 alpha mediated pathogenesis.

Biochemical and Biophysical Research Communications
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1090-2104; 0006-291X