Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture

Jung, Jae-Wan; Kim, Nan-Sun; Jang, Seon-Hui; Shin, Yun-Ji; Yang, Moon-Sik

doi:10.1016/j.jbiotec.2016.03.031

Cited by 36 publications

(21 citation statements)

References 49 publications

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“…The presence of these structures suggests that, despite low detection of rrBChE in the culture medium, the molecule is indeed being processed through the secretory pathway as intended. The structure, relative abundance, and heterogeneity of these glycans across the sites match what has been previously reported for recombinant proteins secreted from rice cell suspension cultures (Jung, Kim, Jang, Shin, & Yang, ; Kang et al, ), and are similar to those secreted from whole Nicotiana benthamiana plants (Alkanaimsh et al, ; Schneider, Marillonnet et al, ). Glycans terminating in galactose residues are likely a result of incomplete formation of the LewisA structure, which is commonly found in the rice secretome (Jung et al, ), though not always detected in high abundance (Kang et al, ; Shin, Chong, Yang, & Kwon, ).…”

Section: Resultssupporting

confidence: 81%

Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

Corbin

Kailemia

Cadieux³

et al. 2018

Biotech & Bioengineering

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Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use.

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Section: Resultssupporting

confidence: 81%

Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

Corbin

Kailemia

Cadieux³

et al. 2018

Biotech & Bioengineering

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“…In recent years, several lysosomal enzymes for therapeutic use in ERT have been successfully produced using plant-based expression platforms [12][13][14][15] (see [16] for review). GAA has been effectively produced in several plant-hosts, e.g., in rice calli [17] and tobacco [18]. Plant-based systems in general profit from advantages like lower production costs, eliminated risk of contamination by zoonotic pathogens and improved process stability.…”

Section: Introductionmentioning

confidence: 99%

Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease

Hintze

Limmer

Dabrowska‐Schlepp³

et al. 2020

IJMS

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Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa−/− mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

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“…결과적으로 목적생 산물의 in situ 회수를 통해 하류 공정의 단계도 줄이면서 부 피당 생산성까지 증가시키는 효과를 얻을 수 있다. 형질전환 식물세포를 이용한 재조합 단백질 생산은 세포의 내부에 단백질을 축적해 생산하거나, 세포 외부로 단백질을 분비하여 생산한다 [9,10]. 세포 외부로의 재조합 단백질의 분비는 세포를 파쇄하지 않고 배지 내 단백질의 분석과 정제 를 할 수 있기 때문에 생물공정에 있어 유리하다 [11].…”

Section: Introductionunclassified

In situ Recovery of hCTLA4Ig from Suspension Cell Cultures of Oryza sativa

Yeol¹,

전수환²,

권준영³

et al. 2016

KSBB Journal

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In this research, recombinant human cytotoxic Tlymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced by transgenic rice cells. RAmy3D promoter was used for overcome the limitation of low expression level in transgenic plant cells, and the secretion of target protein was accomplished by signal peptide. However, the RAmy3D promoter system which can be induced only by sugar starvation causes the decrease of cell viability. As a result, cell death promotes the release of protease which degrades the target proteins. The protein stability and productivity can be significantly influenced by proteolysis activity. Therefore, development of new strategies are necessary for the in situ recovery of target proteins from cell culture media. In this study, in situ recovery was performed by various strategies. Direct addition of Protein A resin with nylon bag leads to cell death by increased shear stress and decrease in production of hCTLA4Ig by protease. Medium exchange through modified flask could recover hCTLA4Ig with high cell viability and low protease activity, on the other hand, the productivity was lower than that of control. When in situ recovery was conducted at day 7 after induction in air-lift bioreactor, 1.94-fold of hCTLA4Ig could be recovered compared to control culture without in situ recovery. Consequently, in situ recovery of hCTLA4Ig from transgenic rice cell culture could enhance productivity significantly and prevent degradation of target proteins effectively.

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Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture

Cited by 36 publications

References 49 publications

Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

Purification, characterization, and N‐glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures

Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease

In situ Recovery of hCTLA4Ig from Suspension Cell Cultures of Oryza sativa

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