Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease

Tobacco phytaspase: Successful expression in a heterologous system

“…The protein was subsequently purified by affinity chromatography using glutathione-agarose beads as described previously. 18 Consistent with our results expressing GST-mature phytaspase in our recent study, we were successful in obtaining GST-pre-prophytaspase in a soluble fraction, though with very low expression (Fig. 2b).…”

“…We have recently demonstrated the successful expression of mature phytaspase (without the pro-domain) from tobacco in a bacterial system. 18 Despite our success and other reports of the effective expression of active recombinant mature subtilases without the pro-domain, we cannot reject the possibility that more active phytaspase can be obtained when expressed with the prodomain. Therefore, we focus on the bacterial expression of pre-prophytaspase in the present communication.…”

“…Our recent study on GST-mature phytaspase has demonstrated that the mature phytaspase without any pro-domain maintains its catalytic activity once the GST-tag is removed. 18 We have also been successful in expressing pre-prophytaspase in a bacterial system and have made preliminary observations of the recombinant preprophytaspase. However, like many other research groups working on plant subtilases, 9 we also have failed to obtain this precursor phytaspase in high enough amounts to carry out further investigations.…”

“…10,14,19,20 We cloned the tobacco pre-prophytaspase gene into the pGEM-T Easy vector as described in a previous study. 18 The pGEM-T Easy-pre-prophytaspase construct was used as a template for the further amplification of pre-prophytaspase for subcloning into the pET-28a and pGEX-4T2 vectors (Fig. 1).…”