Expression, purification and biophysical characterization of recombinant Streptomyces violaceoruber phospholipase PLA2 overproduced in Pichia pastoris
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… The PLA2 gene sequence was synthesized using GenScript (USA) Plasmids, strains and culture media … The codon-optimized PLA2 gene for P pastoris expression from S violaceoruber (Sequence ID AY3598661, 363 bp) was synthesized (GenScript, USA) … |
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Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73?U/mg was purified with a concentration of at least 3?mg/mL. T... More
Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73?U/mg was purified with a concentration of at least 3?mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca2+ and Ba2+ ions significantly increased thermostability of the enzyme.
Methylotrophic yeast; Pichia pastoris; phospholipase A2
