Arabidopsis Hairy Roots Producing High Level of Active Human Gastric Lipase
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ORIGINAL PAPER
Arabidopsis Hairy Roots Producing High Level of Active Human Gastric Lipase François Guerineau1 · Nga T. P. Mai1,2 · Michèle Boitel‑Conti1
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Arabidopsis hairy roots were used to produce human gastric lipase. When treated with 2,4-D, the hairy roots developed into thick organs that produced more protein than untreated roots. This was first assessed using green fluorescent proteinproducing root lines from which the protein diffused into the culture medium. When growing hairy roots which express the human gastric lipase gene, very little lipase was found in the medium. Incubating the roots in a low pH buffer resulted in lipase diffusion into the buffer, avoiding the need for grinding. The activity of the enzyme on 4-methylumbellireryl-oleate and on tributyrin was determined. Approximately 6000 units of enzyme were recovered per gram of root. The enzyme was also extracted from freeze-dried roots before and after a 2-month storage period at room temperature. This work demonstrates the relevance of Arabidopsis hairy roots for the production of human gastric lipase. Keywords Auxin · 2,4-D · Arabidopsis · Gastric lipase · Hairy roots Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BCA Bicinchoninic acid BSA Bovine serum albumin hGL Human gastric lipase MU 4-Methylumbellirerone MUO 4-Methylumbellireryl-oleate
Introduction Several approaches have been explored for the production of heterologous proteins in plants: production in whole plants, seeds or in plant tissues or cells grown in vitro [1–4]. The regulatory issues with field-grown and fertile transgenic plants led to the development of transient expression techniques which can be used on plants grown in greenhouses or growth chambers, thus not generating transgenic pollen or * François Guerineau francois.guerineau@u‑picardie.fr 1
Biologie Des Plantes Et Innovation (BIOPI), Université de Picardie Jules Verne, 33 rue St Leu, 80039 Amiens, France
Present Address: University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay District, Hanoi, Vietnam
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seeds [5, 6]. Transient expression requires fully-grown plants and bacterial cultures so that leaves can be infiltrated with Agrobacterium, which will transfer the foreign gene to many leaf cells. A few days later, leaves are harvested. Obtaining a pure protein therefore requires leaf grinding, bacterial elimination and extensive protein purification to separate the protein of interest from the plant proteins. The main advantage of transient expression is speed: the production takes a few days from the time plants are ready to be infiltrated. Because the expression is taking place in leaves growing on whole plants, the level of production can be affected by environmental factors, or compromised by pathogen attacks [7]. In contrast, the in vitro techniques involve growing transgenic plant cells or tissues under aseptic conditions. The producti
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