Molecular cloning, overexpression and characterization of human interleukin 1alpha.
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.
Rajalingam
Dakshinamurthy
D
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Kacer
Doreen
D
Prudovsky
Igor
I
Kumar
Thallapuranam Krishnaswamy Suresh
TK
D002151:Calorimetry / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000187:drug effects; D006801:Humans; D053582:Interleukin-1alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009418:S100 Proteins / Q000378:metabolism
D053582:Interleukin-1alpha; D011994:Recombinant Proteins; D009418:S100 Proteins; C511523:S100A13 protein, human