An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

Abstract

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A 2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A 2 specific activities evidence the homogeneity of the isolated proteins.

Different types of aqueous two‐phase systems for biomolecule and bioparticle extraction and purification

“…Several therapeutic proteins were studied using the ATP system, mostly antibodies produced in different sources such as in mammalian cells, yeast, plants, transgenic sheep milk, and bacteria . Other biomolecules of promising therapeutic interest were also studies as phycobiliproteins or protease and phospholipase from venom . ATPS antibodies extractions were extensively studied in different systems, achieving different results; some of which will be further discussed.…”

Primary recovery strategies of low‐molecular‐weight toxins from Crotalus molossus nigrescens and Crotalus atrox using aqueous two‐phase and three‐phase partition systems

“…Only polyethylene glycol (PEG)-phosphate, PEG-sulfate, and alcohol-salt systems have been tested for the partition of proteases and phospholipases A 2 from Bothrops alternatus, B. jararacussu, and Crotalus molossus nigrescens (CMN) venoms. [17][18][19][20] On the other hand, TPP is a fast and easy separation method that allows concentration and partial purification of different molecules. 21 Three-phase separation occurs when the biological sample is mixed with ammonium sulfate, at a given saturation percentage, and t-butanol.…”

“…Although several molecules from different sources have been recovered using ATPS, its use for snake venom fractionation has been poorly explored. Only polyethylene glycol (PEG)–phosphate, PEG–sulfate, and alcohol–salt systems have been tested for the partition of proteases and phospholipases A 2 from Bothrops alternatus, B. jararacussu , and Crotalus molossus nigrescens (CMN) venoms 17‐20 …”

Development of aqueous two-phase systems-based approaches for the selective recovery of metalloproteases and phospholipases A2 toxins from Crotalus molossus nigrescens venom

“…While ATPS are widely used for recovering proteins from complex matrices, application for recovering proteins from venoms is uncommon. To our knowledge, only three other studies have reported the Enriquez-Ochoa et al Bioresources and Bioprocessing (2021) 8:136 use of this technique for the isolation of PLA 2 and MPs from snake venoms (Gómez et al 2012;Da Silva et al 2015;Gomez et al 2016). The large variation of venoms composition and activities, both at intra and interspecies levels, makes necessary the establishment of recovery conditions based on particular snake species from a certain region.…”