| Nitric oxide has been found to be an important biomessenger and bioregulator in the nervous, immune and cardiovascular systems. Since it carries such important roles in an organism, understanding the regulation of NO is important. While the mechanism of constitutive nitric oxide synthases has been widely understood, the regulation of iNOS has been unclear. There are several attempts trying to model the iNOS holoenzyme-CaM complex using several approaches such as kinetic, proteolysis, mass spectroscopy or circular dichroism. The previous studies provide valuable results including partial binding sites of NOS/CaM complex and the orientation of these two molecules. The ultimate goal of this project is to identify the binding sites on iNOS and CaM including intramolecular and intermolecular binding sites, and to combine the results from these experiments and from previous studies to simulate the binding pattern between iNOS and CaM. However, due to the difficulty of acquiring the sufficient amount of purified iNOS/CaM complex to achieve that goal. The purification of the iNOS/CaM complex from 4L BL21 (DE3) culture had been performed on 2', 5' --ADP Sepharose, Ni2+ NTA, and S200 gel filtration column. The purified active iNOS had been collected from only Ni2+ NTA column with 89% total iNOS yield and 200-fold purification. However, S200 gel filtration is required to achieve the more purified iNOS/CaM. By up scaling 4L BL21 (DE3) to 8L, the amount of the purified iNOS from Ni2+ NTA will be enough to applied to S200 gel filtration column. The restriction digestion of iNOS/CaM confirmed the CaM gene insert on pCACYC184 and confirmed 5' NdeI restriction site but did not confirm 3' XbaI site. |
