Trans-activation of recombinant haloalkaliphilic protease produced in E. coli

DIEGO M. RUIZ; MARÍA I. GIMÉNEZ; ROSANA E. DE CASTRO

Rosario

Congreso; V Congreso Argentino de Microbiología General (SAMIGE); 2008

Sociedad Argentina de Microbiologia General (SAMIGE)

Optimization of heterologous production of enzymes with potential use in Biotechnology –such as proteases- is highly desirable. Enzymes from extremophilic organisms are active in conditions where their mesophilic counterparts are denatured or inactivated, which makes them a very interesting tool for biotechnological processes. The haloalkaliphilic archaeon Natrialba magadii produces an extracellular serine protease (Nep) that has been characterized in our laboratory. Nep is closely related to several proteases of the subtilisin family. Enzymes of this family are synthesized as inactive precursors with a signal peptide followed by a propeptide at the N-terminus. Once translocated, the pre-peptide is cleaved by a signal peptidase and then the propeptide is removed autoproteolitically.The nep gene was cloned and expressed in two different organisms: the haloneutrophilic archaeon Haloferax volcanii and Escherichia coli. The recombinant enzyme produced in Hfx. volcanii was identified as Nep (HvNep) by mass spectrometry and had similar biochemical properties as the native protease. However, purification of the recombinant enzyme by bacitracine or nickel affinity chromatography was not successful.The recombinant protease produced in E. coli cells (EcNep) was identified as the precursor of Nep by mass spectrometry and these cell extracts showed very low activity. Unprocessing of pre-pro-Nep in E. coli may be due to the low salt conditions preventing proper folding and thus further processing/translocation of the protease; lack of the components necessary for these processes or a combination of both factors.In this work we show that adding small amounts of native mature Nep to cell extracts of recombinant E. coli expressing pre-pro-Nep-His6 triggers protease activation. This process is dependent on time, temperature and amount of protease added in trans. Moreover, pre-proNep-His6 was purified by nickel affinity chromatography and activated by addition of mature Nep. The affinity purified polypeptides had similar electrophoretic mobility as the protein species identified as pre-pro-Nep in E. coli cell extracts. Preliminary results show that trans-activated EcNep cannot be purified by nickel affinity chromatography suggesting that the His6 tag may be removed by autoproteolysis. This could be a possible explanation for our previous results with HvNep.Taken together, these results suggest that the processing of Nep is autoproteolytic and that the factors and/or conditions necessary for triggering protease activation are absent in E. coli cells. In addition, trans-activation of EcNep opens the possibility of large scale production of active recombinant enzyme for future applications.This work was supported by UNMdP, ANPCyT y CONICET.