Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Abstract

Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

“…Together, our results revealed that Cdtsp 2 has a high degree of structural similarity to other snake venom serine proteases, including the presence of a hydrophobic cavity on its surface, and the conservation of amino acid residues involved in the activity of snake venom serine protease, such as H43, H56, D92, and S188 (Figure 2) [11,28]. These conserved amino acid residues are located between the N-terminal and C-terminal regions, which features a hydrophobic cavity typical of major serine proteases, containing the catalytic triad of residues [2,5,16,28] (Figure 1 and Figure 2B). A study on serine proteases isolated from Lachesis muta revealed that the residues, F86, W89, Y160, F203, and W204, form an essential hydrophobic cavity involved in binding fibrinopeptide A and chromogenic substrates [22].…”

“…Molecular modeling and structural comparisons of this simile gyroxin protein with other highly conserved serine proteases revealed that this gyroxin simile isolated from Crotalus durissus collilineatus comprised several common domains found in various thrombin-like enzymes of other poisons [1,2]. In addition, serine proteases isolated from Crotalus durissus cumanensis venom also showed the presence of two serine-protease fractions through a combination of molecular exclusion on a Sephacryl S-200 column and reverse-phase HPLC on a semi-preparative C8 column [3].…”

“…These classes of toxins represent an essential tool for the capture of prey using envenomation, on the basis of pivotal toxins being responsible for several blood homeostasis disorders during a snake venom bite, leading to major toxicity for some species. Since SVSPs are proteins that modulate blood homeostasis, several studies revealed their potential therapeutic use in the treatment of blood coagulopathy [2,4,5]. All SVSPs identified to date present a catalytic triad comprising histidine (His), aspartate (Asp), and serine (Ser), essential for their enzyme activity [2,5].…”

“…Since SVSPs are proteins that modulate blood homeostasis, several studies revealed their potential therapeutic use in the treatment of blood coagulopathy [2,4,5]. All SVSPs identified to date present a catalytic triad comprising histidine (His), aspartate (Asp), and serine (Ser), essential for their enzyme activity [2,5]. Recent studies strongly suggested that serine proteases BpirSP27 and BpirSP41, purified from Bothrops pirajai (Bpir), were able to activate the complement system, potentially inducing edema under experimental conditions, and may include proinflammatory features, as well as being significantly inhibited by nonsteroidal anti-inflammatory drugs [6,7,8].…”

Beyond hemostasis: a snake venom serine protease with potassium channel blocking and potential antitumor activities

“…Collinein-1 is a 29.5 kDa thrombin-like serine protease isoform from C. d. collilineatus venom that cleaves preferentially the Aα chain of fibrinogen 23 . The recombinant form of collinein-1 (rCollinein-1) was previously produced with functional integrity, using the Pichia pastoris heterologous expression system 23,24 . The screening of collinein-1 activity on voltage-gated ion channels was performed with rCollinein-1 (5 μM) on different isoforms of voltage-gated potassium (Kv) and sodium (Nav) channels expressed in Xenopus laevis oocytes.…”