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1.
Front Bioeng Biotechnol ; 10: 838365, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35252146

RESUMO

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with ß1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human ß1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric ß1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human ß1,4-galactosyltransferase. Stable FTGT expression led to the desired ß1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of ß1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

2.
Front Plant Sci ; 11: 610032, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33391325

RESUMO

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of ß1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human ß1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous ß1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

3.
Mol Biosyst ; 10(7): 1679-88, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24469598

RESUMO

On command control of gene expression in time and space is required for the comprehensive analysis of key plant cellular processes. Even though some chemical inducible systems showing satisfactory induction features have been developed, they are inherently limited in terms of spatiotemporal resolution and may be associated with toxic effects. We describe here the first synthetic light-inducible system for the targeted control of gene expression in plants. For this purpose, we applied an interdisciplinary synthetic biology approach comprising mammalian and plant cell systems to customize and optimize a split transcription factor based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). Implementation of the system in transient assays in tobacco protoplasts resulted in strong (95-fold) induction in red light (660 nm) and could be instantaneously returned to the OFF state by subsequent illumination with far-red light (740 nm). Capitalizing on this toggle switch-like characteristic, we demonstrate that the system can be kept in the OFF state in the presence of 740 nm-supplemented white light, opening up perspectives for future application of the system in whole plants. Finally we demonstrate the system's applicability in basic research, by the light-controlled tuning of auxin signalling networks in N. tabacum protoplasts, as well as its biotechnological potential for the chemical-inducer free production of therapeutic proteins in the moss P. patens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Nicotiana/genética , Fitocromo B/metabolismo , Animais , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células CHO , Cricetulus , Vetores Genéticos/genética , Luz , Fitocromo B/genética , Plantas Geneticamente Modificadas , Protoplastos/metabolismo , Biologia Sintética
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