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Production and characterization of recombinant restriction enzyme NgoMIV and its mutant

Abstract

Discovery of restriction enzymes has led to the development of recombinant DNA technology and restriction enzymes are currently widely used in biotechnology, medicine and research. Till now, thousands of restriction enzymes have been discovered, purified, characterized and commercialized. As well as, production of restriction enzymes has gained a huge profit in biotechnology industry. Although restriction enzymes have been applied well in current molecular biology researches, a lot of questions regarding their binding and catalytic mechanisms haven't been clearly explained or described by scientists. NgoMIV is a tetrameric restriction enzyme, which has been discovered and purified about 20 years ago from Neisseria gonorrhoeae MS11. It belongs to the type II P and type II F subtypes. The recognition site of NgoMIV is 5'-G/CCGGC- 3', which is a palindromic sequence. Also the tetrameric protein has two catalytic cores, which means that it will bind with two cognate sites and cut them both. NgoAIV, which was discovered in another strain- FA1090 of Neisseria gonorrhoeae, is different from NgoMIV protein in two amino-acid residues. A hybrid protein called NgoIV-hb was designed based on the differences. In the thesis study here, the expression of NgoMIV and its mutant was conducted. To undercover the preference of the distance between two recognition sites, digestion tests by applying a plasmid with four recognition sites as substrate were conducted. At last, several proposed mechanisms of catalysis based on the protein motion predicted by anisotropic elastic network model and current existing crystal structures were raised

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