Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube.
The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.
Li
Rong-Gui
RG
Department of Biology, Qingdao University, Qingdao 266071, China.
Qian
Dong-Meng
DM
Guo
Dao-Sen
DS
Du
Gui-Cai
GC
Yan
Zhi-Yong
ZY
Wang
Bin
B
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D015801:Helminth Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010960:Plasminogen Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011077:Polychaeta / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014431:Tylenchoidea / Q000201:enzymology
D002364:Caseins; D018076:DNA, Complementary; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D010960:Plasminogen Activators; D012697:Serine Endopeptidases