Characterization, Expression And Functional Analysis Of Elastase Gene In Amphioxus Branchiostoma Belcheri Tsingtauense

Elastase (ELAS) belongs to the family of serine proteinase, and it is unique among proteinases in having the ability to rapidly hydrolyse Elastin . It is also able to hydroly -se various other proteins like haemoglobin, casein and fibrin. ELAS is widely distribut -ed in organisms ranging from invertebrates to vertebrates. Little is known to date about ELAS in the amphioxus Branchiostoma belcheri. Amphioxus or lancelet, a cepha -lochordate, has long been regarded as the living invertebrate most closely rElasted to the proximate invertebrate ancestor of vertebrates. In this paper, we report expression and functional analysis of ELAS gene in amphioxus B. belcheri tsingtauense.The cDNA encoding B. belcheri ELAS, BbElas, obtained from the gut cDNA library of Branchiostoma belcheri is 987 bp long. Its longest ORF encods a 277 amino acids long protein with a predicted molecular mass of approximately 29 kDa, and it has a 16 amino acids long N-terminal signal peptide. Like other Elas, BLASTp searching at NCBI showed that the deduced protein BbElas had a Tryp_SPc domain at residual positions 31-267, with a highly conserved sequence GDSGGPL and a catalytic triad His74- Asp127-Ser222, and eight cysteine residues at positions 59, 75, 161, 191, 207, 218, 228 and 248, that are typical of Elastases. Alignment of with Elas of other species revealed that the BbElas clustered with the Elas of invertabrates and at the root of vertabrates, conforming the important position of Amphioxus in evolution.Northern blotting, Insitu hybridization histochemistry demonstrated that BbElas transcript was abundant in the mid-gut. For investigating the function of BbElas, the recombinamt BbElas has been constructed, transfered to E.coli and induced to expressed. BbElas expressed in insoluble body with a abundant amount. We denatured the insoluble body of BbElas amd purified it. Using the Elastase specific substrate N-Succinyl-Ala-Ala-Ala-p- nitroanilide we detected the enzyme activity of recombinamt BbElas, and got an enzyme activity of 0.22±0.02 U/mg;After that,the effect of some protein inhibitors on the recombinamt BbElas to N-Succinyl-Ala-Ala-Ala-p- nitroanilide was studied, and it shows that PMSF and Elastinital, the inhibitor special to the serine proteinase and Elastase, have a strong inhibition to the renatured protein, and that further indicates the recombinamt BbElas is a kind of Elastases belonged to the serine proteinase superfamily.