Hairy-root organ cultures for the production of human acetylcholinesterase

Ryan R Woods,Tsafrir S Mor,Brian C Geyer

doi:10.1186/1472-6750-8-95

Abstract

BackgroundHuman cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.ResultsAs a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4°C for up to 5 months.ConclusionOur results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.

Highlights

Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents
Creation of hairy root cultures Previously we described the creation of transgenic N. benthamiana plants expressing the "readthrough" isoform
Thirteen independent hairy root lines were generated by A. rhizogenes infection and screened for the presence of the transgene and for levels of recombinant protein, assayed by its enzymatic activity (Fig. 1b)

Summary

Introduction

Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified. Bioscavenging of organophosphate (OP) by human cholinesterases (ChEs) is emerging as a promising medical intervention for prophylaxis and post-exposure treatment against chemical warfare nerve agents and pesticides, meeting considerable success in pre-clinical studies [1,2]. ChEs are very efficient in sequestering OPs that become esterified to a serine residue at the active site. Of the two ChEs in humans, only the serum enzyme butyrylcholinesterase (BChE) can be obtained from natural sources, and large-scale purification efforts from outdated blood-banked human plasma were demonstrated (page number not for citation purposes)

Methods

Results

Conclusion