Dataset Information

Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

ABSTRACT: To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed.

SUBMITTER: Alkanaimsh S

PROVIDER: S-EPMC4909763 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

Alkanaimsh Salem S Karuppanan Kalimuthu K Guerrero Andrés A Tu Aye M AM Hashimoto Bryce B Hwang Min Sook MS Phu My L ML Arzola Lucas L Lebrilla Carlito B CB Dandekar Abhaya M AM Falk Bryce W BW Nandi Somen S Rodriguez Raymond L RL McDonald Karen A KA

Frontiers in plant science 20160616

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to ...[more]

PMID: 27379103

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Project description:BackgroundAgroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis.ResultsUsing the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1-2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform.ConclusionsIn this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

Project description:Interest in the plant-based transient production of recombinant immunogenic antigens has tremendously progressed because plants are cost-effective, easily selectable, free of mammalian contamination, and support complex post-translational modifications. Nicotiana benthamiana is a convenient system for transient expression of recombinant antigens. The present study documented a platform for rapid production of Helicobacter pylori CagA, VacA and NapA antigens three days (first harvest, FH) and six days (second harvest, SH) after agro-infiltration using a syringe. In this study, CagA, VacA and NapA antigen genes from Helicobacter pylori were cloned into the binary vector pBI121 and transformed into Nicotiana benthamiana by the Agrobacterium-mediated process. Leaves of four to five weeks old Nicotiana benthamiana plants were agroinfiltrated with EHA105 subtype of Agrobacterium tumefaciens strain containing cloned CagA (pBI121-CagA), VacA (pBI121-VacA) and NapA (pBI121-NapA) constructs. The transient expression and accumulation of the recombinant genes containing CagA, VacA and NapA expression cassettes were confirmed using qRT-PCR by comparing the relative expression at FH and SH post-infiltration with the non-infiltrated (control) samples and using ELISA at 1/5 and 1/10 dilution ratios. The qRT-PCR findings showed that Agrobacterium-mediated syringe infiltration of leaves of four to five weeks old Nicotiana benthamiana plants produced significantly higher transcript levels of CagA (about 8-fold and 7-fold), VacA (38-fold and 24-fold) and NapA (7-fold and 5-fold) genes at FH and SH compared to the control sample. Besides, the maximum amount of CagA, VacA and NapA antigens were detected at the FH stage compared to the SH stage, when the antibody concentrations of the agro-infiltrated leaf extracts containing these recombinant antigens were diluted in a 1/5 ratio. This study has developed evidence to support that recombinant CagA, VacA and NapA can be transiently produced in Nicotiana benthamiana plants.

Dataset Information

Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

Publications

Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

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