Molecular cloning, overexpression and characterization of human interleukin 1alpha |
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Authors: | Rajalingam Dakshinamurthy Kacer Doreen Prudovsky Igor Kumar Thallapuranam Krishnaswamy Suresh |
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Institution: | Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. |
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Abstract: | Interleukin-1 alpha (IL-1alpha) 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-1alpha. Human IL-1alpha has been expressed in Escherichia coli in high yields ( approximately 4mg per liter of the bacterial culture). The protein was purified to homogeneity ( approximately 98% purity) using affinity chromatography and size exclusion chromatography. Results of the steady-state fluorescence and 2D NMR experiments show that the recombinant IL-1alpha is in a folded conformation. Far-UV circular dichroism (CD) data suggest that IL-1alpha is an all beta-sheet protein with a beta-barrel architecture. Isothermal titration calorimetry (ITC) experiments show that the recombinant IL-1alpha binds strongly (K(d) approximately 5.6 x 10(-7) M) to S100A13, a calcium binding protein that chaperones the in vivo release of IL-1alpha into the extracellular compartment. Recombinant IL-1alpha 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-1alpha but also provide avenues for the rational design of potent inhibitors against IL-1alpha mediated pathogenesis. |
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Keywords: | IL-1α interleukin-1 alpha NMR nuclear magnetic resonance spectroscopy CD circular dichroism ITC isothermal titration calorimetry SAR structure and activity relationship HSQC hetero nuclear single quantum coherence ER endoplasmic recticulum HUVEC human umbilical vein endothelial cells MALDI matrix assisted laser desorption/ionization IPTG d-1-thiogalactopyranoside" target="_blank">isopropyl β-d-1-thiogalactopyranoside |
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