title: Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana.
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, pMAL-c2x, 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.
authors.affiliation: College of Life Sciences, Anhui Agricultural University, Hefei, China.
authors.forename: D H
authors.initials: DH
authors.lastname: Li
authors.affiliation: College of Life Sciences, Anhui Agricultural University, Hefei, China.
authors.forename: H
authors.initials: H
authors.lastname: Xi
authors.affiliation: College of Life Sciences, Anhui Agricultural University, Hefei, China.
authors.forename: X B
authors.initials: XB
authors.lastname: Yu
authors.affiliation: College of Life Sciences, Anhui Agricultural University, Hefei, China.
authors.forename: Y P
authors.initials: YP
authors.lastname: Cai
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004789:Enzyme Activation; D015870:Gene Expression; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D013381:Subtilisins