Bacterial Expression of Human Butyrylcholinesterase as a Tool for Nerve Agent Bioscavengers Development

Abstract

Human butyrylcholinesterase is a performant stoichiometric bioscavenger of organophosphorous nerve agents. It is either isolated from outdated plasma or functionally expressed in eukaryotic systems. Here, we report the production of active human butyrylcholinesterase in a prokaryotic system after optimization of the primary sequence through the Protein Repair One Stop Shop process, a structure- and sequence-based algorithm for soluble bacterial expression of difficult eukaryotic proteins. The mutant enzyme was purified to homogeneity. Its kinetic parameters with substrate are similar to the endogenous human butyrylcholinesterase or recombinants produced in eukaryotic systems. The isolated protein was prone to crystallize and its 2.5-Å X-ray structure revealed an active site gorge region identical to that of previously solved structures. The advantages of this alternate expression system, particularly for the generation of butyrylcholinesterase variants with nerve agent hydrolysis activity, are discussed.

Keywords: 3D structure; PROSS; SEC-MALS; butyrylcholinesterase; differential scanning fluorimetry; prokaryotic expression.

Figure 1

Structural representation of the selected conserved residues of human butyrylcholinesterase (hBChE) for the PROSS process. Residues conserved in order to preserve enzymatic activity are represented in red, those conserved to maintain dimer interaction are represented in cyan.

Figure 2

Comparison of BChE activity in the bacterial soluble extracts of the different BChE constructs. An aliquot on each soluble extract from pThioHis constructs of hBChE-wild-type (WT) and hBChE-1 to hBChE-7 was measured for butyrylthiocholine hydrolase activity. Activities were normalized to the highest measured sample.

Figure 3

Analysis of the purification of hBCHE-7. A polyacrylamide gel electrophoresis was realized in denaturing conditions with an aliquot of different purification steps. Molecular weight (MW, and corresponding digits in kDa); total soluble extracts (TSE); flow-through, wash and elution fractions of the first metal ion affinity chromatography (IMAC1); dialysis and TEV-protease cleavage (TEV); and, flow-through fraction of the second IMAC (IMAC2).

Figure 4

Oligomeric state analysis of pure hBChE-7. After separation of pure hBChE-7 on Superdex 200 Increase 10/300 equilibrated in 20 mM Tris pH 8.0, 150 mM NaCl, the major UV peak (plain line) was analyzed in line by multi-angles light scattering and a constant molecular weight of 70 + 2 kDa was measured (dashed line).

Figure 5

Crystals of hBChE-7. Crystallization was realized by the hanging drop method using 0.2 M NH₄OAc pH 7.4 and 20% polyethylene glycol (PEG) 3350 as precipitation solution. hBhE-7 at a concentration of 2 mg mL⁻¹ was mixed 2:1 with the precipitation solution. (A) crystals grown at 20 °C; (B) crystals grown at 4 °C.

Figure 6

Structure of hBChE-7. (A) cartoon representation of the in cristallo dimer form of hBChE-7 (PDB ID 6EMI) as red and blue chains. Superposition of the structure of recombinant hBChE from CHO cells (PDB ID 1P0I), green chain; (B) representation of the broken Cys65-Cys92 disulfide bond in chain B. Protein backbone is represented in gray. Residues Cys65 and Cys92 are represented as sticks with carbon atoms in gray, oxygen atoms in red, nitrogen atoms in blue and sulfide atoms in yellow. An |F_o − F_c| electron density map calculated by omitting residues Cys65 and Cys92 from the model is represented as a green mesh with a 3.0 σ contour.

Figure 7

Active site gorge comparison of hBChE-7 and recombinant hBChE produced in CHO cells. Residues forming the active site triad (Ser198, Glu335, and His438) and those forming the active-site gorge are represented as sticks. Carbon atoms are represented in green and magenta for hBChE-7 structure (PDB ID 6EMI) and hBChE expressed in CHO cells (PDB ID 1P0I), respectively. Nitrogen atoms are represented in blue and oxygen atoms in red. Water molecules are represented as red spheres. The active site gorge is represented as a semi-transparent grey surface.