The fine transformation between the oxidized and reduced forms of interleukin-1 receptor antagonist

The active and pure recombinant forms of human interleukin-1 receptor antagonist (rHIL-1ra) were obtained from inclusion body expressed in Escherichia coli BL21 (DE3). However, the final purified protein was found to be in an unstable reduced form and easily converted to the other forms. Which form of the protein was involved and precisely how this occurred remained obscure. Therefore, we changed oxidizing and reducing conditions to delineate the subtle change between the reduced and oxidized states of rHIL-1ra based on high-pressure liquid chromatography (HPLC). The fine transformation between the two states was further verified through matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectroscopy. Meanwhile, which form might be beneficial to the protein was also investigated by testing its full bioactivity based upon methyl thiazolyl tetrazolium (MTT) method.