OmpT, located in Escherichia coli (E. coli) outer membrane, is a protease that demonstrates highly substrate specificity. In order to estalish the approaches for expression and refolding of membrane protein OmpT, and examine the demonstrated protease activity of OmpT, the ompT gene was first amplified by PCR and inserted into pET28a (pET-ompT) and introduced by Asp85Ala site-directed mutagenesis to generate mutant Asp85Ala (pET-ompT85). Then, the two recombinant plasmids were transformed into BL21 (DE3), OmpT and the mutant were expressed in the form of inclusion bodies, purified and refolded by N-Dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide (LPS) to the recombinant OmpT was critical to refold its protease activity in vitro. Finally, the ability of the recombinant wide-type OmpT to hydrolyze protamine and rabbit muscle creatine kinase (RMCK) was confirmed by SDS-PAGE, bacteria agglutination and growth curve in contrast to the mutant. The results suggest that the desired recombinant OmpT was obtained, which showed the significant protease activity in the protection of E. coli against protamine in vitro.