Use of E-64 cysteine protease inhibitor for the recombinant protein production in <i>Tetrahymena thermophila</i>

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 degrees C and 38 degrees C. In vitro protease activity analysis using secretome as protease enzyme source from E -64 -supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 mu M concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila . This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation .