Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

Jasmine M. Corbin,Brian A. Roberts,Zachary R. Kyser,Bryce I. Hashimoto,Amy R. Noe,Kalimuthu Karuppanan,Liying Wu,Karen A. McDonald,Raymond L. Rodriguez,Somen Nandi

doi:10.3389/fpls.2016.00412

Abstract

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE.

Highlights

Butyrylcholinesterase (BChE, EC 3.1.1.8) is a native human serine hydrolase enzyme that has been shown to function as a bioscavenger against various organophosphorus nerve agents, with both prophylactic and therapeutic applications (Lenz et al, 2005)
The native human BChE (NCBI NM_000055) coding sequence without the native secretion signal peptide was modified for expression in rice and inserted into a vector containing the rice alpha amylase 3D (RAmy3D) promoter, signal peptide, and terminator sequences using GenScript (GenScript, Piscataway, NJ)
The rice-optimized BChE gene construct was cloned into the RAmy3D (Huang et al, 2001) expression system (Figure 1) and subsequently into A. tumefaciens for gene delivery by cocultivation

Summary

Introduction

Butyrylcholinesterase (BChE, EC 3.1.1.8) is a native human serine hydrolase enzyme that has been shown to function as a bioscavenger against various organophosphorus nerve agents, with both prophylactic and therapeutic applications (Lenz et al, 2005). A major limitation in the production of recombinant BChE is the need to produce tetrameric BChE, which has a significantly longer circulatory half-life than the dimeric or monomeric forms (Duysen et al, 2002). Many of these systems show incomplete tetramerization of the molecule (Huang et al, 2007; Geyer et al, 2010; Brazzolotto et al, 2012; Schneider et al, 2014b). Whole plant systems can avoid this problem, and require specialized facilities to grow and harvest transgenic material or to transiently express foreign proteins

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