Molecular cloning, expression, and anti-tumor activity of a novel serine protease from Arenicola cristata.
Arenicola cristata, a marine annelid, is a well-known and prized traditional Chinese medicine. However, the serine protease gene of A. cristata has not been cloned yet. In this study, a novel protease of A. cristata was cloned, sequenced, and expressed in Escherichia coli, and the functions of this recombinant protease were also investigated. The whole complementary DNA (cDNA) of this novel protease was of 980 bp in length and consisted of an open reading frame of 861 bp encoding 286 aa. Sequence analysis of the deduced amino acid sequence revealed that the protease belongs to the serine protease family. The active enzyme of the proposed A. cristata protease is composed of a signal peptide, a propeptide, and a mature polypeptide. The molecular weight of the recombinant mature protein was ~26 kDa after over-expression in E. coli. The recombinant protein significantly inhibited cell growth and induced cell apoptosis of esophageal squamous cell carcinoma (ESCC) in vitro, and reduced tumorigenicity in vivo. Furthermore, administration of the recombinant protein led to the activation of caspase-9 as well as down-regulation of Mcl-1 and Bcl-2. Taken together, our findings indicated that the recombinant serine protease of A. cristata could inhibit ESCC cell growth by mitochondrial apoptotic pathway and might act as a potential pharmacological agent for ESCC therapy.
College of Pharmacy and Biological Science, Weifang Medical University, Weifang 261053, China.
Chunling
C
Zhao
College of Basic Medicine, Weifang Medical University, Weifang 261053, China juimmu@163.com.
Jiyu
J
Ju
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011077:Polychaeta / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D057057:Serine Proteases
Arenicola cristata; apoptosis; esophageal squamous cell carcinoma (ESCC); mitochondrial pathway; serine protease