cDNA cloning and expression of acutin

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Snake venom contains diverse enzymes with significant biological activities.
• Thrombin-like enzymes are a class of serine proteases found in snake venoms, often involved in hemostasis.
• Acutin is a thrombin-like enzyme isolated from Agkistrodon acutus venom, with its specific properties and sequence previously uncharacterized.

Purpose of the Study:

• To purify and characterize acutin, a thrombin-like enzyme from Agkistrodon acutus venom.
• To elucidate the primary amino acid sequence and structural features of acutin.
• To express recombinant acutin and assess its enzymatic activities.

Main Methods:

• Purification of acutin using multiple chromatographic techniques including DEAE-Sepharose CL-6B, Superose 12 FPLC, and Mono-Q.
• Determination of the N-terminal amino acid sequence and isolation of acutin cDNA via RT-PCR.
• Nucleotide sequencing, homology analysis, recombinant protein expression in E. coli, and enzymatic assays.

Main Results:

• Acutin was successfully purified and its N-terminal sequence determined.
• The complete amino acid sequence of mature acutin (233 amino acids) was elucidated, showing homology to other crotaline snake venom thrombin-like enzymes.
• Deduced catalytic residues (His41, Asp84, Ser179) and disulfide bridges were identified.
• Recombinant acutin was expressed, purified, and demonstrated fibrinogen-clotting and arginine-esterase activities.

Conclusions:

• The study provides the first detailed characterization of acutin, including its full amino acid sequence and predicted structure.
• The enzymatic activities of recombinant acutin confirm its role as a functional thrombin-like enzyme.
• This research contributes to the understanding of snake venom composition and the evolution of proteolytic enzymes.