Harvey Wilgus

Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat β-casein promoter. Milk from transgenic animals contained 0.1–5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

BackgroundHuman butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD50 of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.ResultsSecretion level of the fusion protein produced in vitro in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced in vitro was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). In vitro nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.ConclusionBoth the pharmacokinetic study and the in vitro nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced in vitro and in vivo. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.

The molecular weight of the polypeptide chains of yeast phosphofructokinase

Summary When proteolytic artifacts were prevented, yeast phosphofructokinase was found to contain six polypeptide chains each having a molecular weight of 10 5 .

The alkaline isomerization of lysine-modified ferricytochrome c.

Abstract Both totally guanidinated and totally trifluoroacetylated ferricytochromes c exhibit a pH-dependent displacement of the methionine ligand having an apparent p K within 0.5 pH units of that of the native protein. We propose that the pH dependence of this reaction reflects the concentration of hydroxide anion required to displace the methionine ligand.

On the phosphate content of rat liver phenylalanine hydroxylase purified by hydrophobic chromatography

Abstract Rat liver phenylalanine hydroxylase purified by hydrophobic chromatography has been found to contain significantly less protein-bound phosphate than enzyme purified by more conventional procedures. Studies with purified hydroxylase of defined phosphate content suggest that phosphorylated species of phenylalanine hydroxylase possess a higher affinity for the hydrophobic matrix than does the non-phosphorylated form. This selectivity may account for the lower phosphate content in phenylalanine hydroxylase purified by hydrophobic chromatography.

Calibration mixture for estimation of peptide molecular weight by exclusion chromatography

A procedure is described for the rapid preparation of a partial cyanogen bromide digest of horse heart cytochrome c. This digest serves as a calibration mixture for estimation of peptide molecular weight in the range 500 to 20,000 by exelusion chromatography in denaturing solvents. The elution profile of the calibration mixture from Sephadex G-50 equilibrated and developed with 10% formic acid is described by the relationship log M = 4.79 − 0.42 (VoVo).

Biopterin metabolism and phenylalanine hydroxylase activity during early liver regeneration.

Abstract Rat liver biopterin content and the activities of two enzymes involved in biopterin metabolism, sepiapterin reductase and dihydropteridine reductase, were not altered twenty-four hours after partial hepatectomy. This surgical procedure did, however, produce a vigorous regenerative response as verified by an increase in ornithine decarboxylase activity. The tetrahydrobiopterin-dependent activity of phenylalanine hydroxylase was increased in homogenates of regenerating liver. The pteridine requirements for the expression of this activation, and the behavior of the enzyme on calcium-phosphate cellulose columns suggest that elevated levels of cyclic adenosine monophosphate in regenerating liver induce phosphorylation and activation of phenylalanine hydroxylase. This increase in the activity of the primary enzyme of phenylalanine catabolism was interpreted as a compensatory response designed to maintain homeostasis prior to liver regeneration.