Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana

Abstract

ABSTRACT. The Arabidopsis thaliana genome encodes 56 subtilisin-like serine proteases (subtilases). In order to evaluate the protease activity of a previously uncharacterized subtilase, designated as AtSBT1.9, we cloned its full-length cDNA from A. thaliana seedlings. An AtSBT1.9 mature peptide coding sequence was inserted into the bacterial expression vector, pMALc2x, and the recombinant vector was transformed into Escherichia coli BL21 (DE3). The recombinant AtSBT1.9 tagged by maltose binding protein (MBP) was induced as a 117.5-kDa protein in the soluble form in E. coli BL21 (DE3). MBP-AtSBT1.9 was expressed at a level of 11% (w/w) of the bacterial total protein. Protein purification using Amylose Resin revealed a recombinant AtSBT1.9 protease activity of 9.23 U/mg protein at pH 7 and 25°C. Maximal activity occurred over a broad pH (7-8) and temperature (25°-42°C) optimal range. Validation of AtSBT1.9 protease activity would help in characterizing its in vivo function in A. thaliana.

The fungal subtilase AsES elicits a PTI‐like defence response in Arabidopsis thaliana plants independently of its enzymatic activity

“…A,B and Table ), eliminating the autolysis reported for other subtilases (Bajorath et al ., ,b), but also the activity of other proteases present in the periplasmic extract that may degrade the target protein (Zhu et al ., ). Heterologous expression of functional eukaryotic subtilases was previously successfully achieved in E. coli , for example BAJ93208, a barley subtilase that suffers self‐processing when expressed in E. coli (Plattner et al ., ) or AtSBT1.9, one of the many subtilases present in Arabidopsis (Li et al ., ).…”