Production of synthetically created phospholipase A₂ variants with industrial impact

Abstract

Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli. While expression of the gene with an N-terminal leader sequence to direct the protein into the periplasm failed, four variants with slightly modified N-termini (I1A-PLA(2), I1V-PLA(2), His(6)-tagged PLA(2) and PLA(2) still containing the start methionine) were successfully expressed. In all cases, the PLA(2) variants were produced as inclusion bodies. Their protein content amounted to 26-35% of total cell protein. The optimized renaturation procedure and subsequent purification by cation-exchange chromatography yielded pure active enzymes in yields of 4-11 mg L(-1). The recombinant PLA(2) variants showed activities, far-UV CD and fluorescence spectra similar to the glycosylated PLA(2) isolated from the venom glands of honey bee (bv-PLA(2)). The thermodynamic stabilities of the recombinant enzymes calculated from the transition curves of guanidine hydrochloride induced unfolding were also nearly identical to the stability of bv-PLA(2). For the variant I1A-PLA(2) high-cell density fermentation in 10 L-scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably.

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2

“…In our studies on atPLA 2 , bvPLA 2 and ppPLA 2 we noticed significantly different stabilities despite their common fold [26]. In contrast to bvPLA 2 [5,27], which displayed typical cooperative unfolding transitions, ppPLA 2 showed much broader transitions at higher denaturant concentrations or temperatures. The present study aims to reconcile these contradictory stability data of ppPLA 2 .…”

“…In our previous studies of the related bee venom enzyme, bvPLA 2 , we observed, however more consistent parameters (31.4 kJ mol −1 and 3.25 M, respectively) [27]. In order to clarify this apparent inconsistency, we restudied GdnHCl induced unfolding of ppPLA 2 by far-UV CD and intrinsic fluorescence measurements.…”

The role of conformational flexibility on protein supercharging in native electrospray ionization

“…[56][57][58] For example, there is no change in the optical rotary dispersion (ORD) spectrum of honeybee PLA 2 from 10 to 90 1C 59 and no change in the ORD or CD spectra of porcine PLA 2 upon increasing the urea concentration from 0 to 8 M. 60 Common among the members of the PLA 2 superfamily of enzymes 61 are a large number of disulfide bonds (5)(6)(7)(8) for their relatively small sizes (13-18 kDa). [62][63][64] The high density of disulfide bonding is the foundation of their rigid structures and their stability to proteolysis and resistance to denaturation. 57,58 Nanoelectrospray mass spectra of phospholipase A 2 from cobra venom (c-PLA 2 ; 7 disulfide bonds) in aqueous 200 mM ammonium acetate (pH 7.2) solutions containing 0 to 1.5% m-NBA are shown in Fig.…”

Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A2 of Streptomyces violaceoruber

“…Previous studies have been mainly focused on expression and characterization of eukaryotic secretory PLA 2 s. While eukaryotic PLA 2 s have been successfully expressed in yeast [4][5][6][7] and fungus [8], inclusion bodies were formed when expressed in Escherichia coli due to the presence of five to eight disulfide bonds [9,10]. Nevertheless, it would be desirable to establish an E. coli-based PLA 2 expression system because eukaryotic systems are generally considered as time consuming and uneconomic in comparison to prokaryotic systems.…”