Low Protease Content in <i>Medicago truncatula</i> Cell Cultures Facilitates Recombinant Protein Production

Santos, Rita B.; Chandrasekar, Balakumaran; Mandal, Manoj Kumar; Kaschani, Farnusch; Kaiser, Markus; Both, Leonard; Hoorn, Renier A. L. van der; Schiermeyer, Andreas; Abranches, Rita

doi:10.1002/biot.201800050

Cited by 17 publications

(11 citation statements)

References 50 publications

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“…Consequently, searching for plant species with low proteolytic activity will provide improved recombinant protein stability and the screening of plant species with low protease activity is, therefore, urgently required. In fact, Santos et al [154] showed that the number of proteases found in Medicago was considerably lower. Genomic and proteomic approaches will allow us to advance in the knowledge of the specific role of the different proteases and their inhibitors and this will result in the development of new strategies to improve plant-based recombinant protein production significantly.…”

Section: Challenges and Perspectives In Preventing Proteolysis In mentioning

confidence: 99%

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

Clemente

Corigliano

Pariani

et al. 2019

IJMS

127

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The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

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Section: Challenges and Perspectives In Preventing Proteolysis In mentioning

confidence: 99%

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

Clemente

Corigliano

Pariani

et al. 2019

IJMS

127

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“…Although its role is still unknown, the fact that the granulin-domains were uncovered may help explain a possible interaction between the cysteine proteases and cardosin B. In human cells, progranulin has been found to interact with both prosaposin 29 , a glycoprotein involved in the lysosomal trafficking pathway, and cathepsin D, a human aspartic protease 32,33 ). Like most typical plant APs, cardosin B contains a saposin-like domain corresponding to the PSI segment.…”

Section: Discussionmentioning

confidence: 99%

Expression of recombinant cardosin B in tobacco BY2 cells: an alternative system for the production of active milk clotting enzymes

Folgado

Abranches

2021

Preprint

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Cynara cardunculus L. or cardoon is a plant that is used as a source of milk clotting enzymes during traditional cheese manufacturing. This clotting activity is due to aspartic proteases (APs) found in the cardoon flower, named cyprosins and cardosins. APs from cardoon flowers display a great degree of heterogeneity, resulting in variable milk clotting activities and directly influencing the final product. Producing these APs using alternative platforms such as bacteria or yeast has proven challenging, which is hampering their implementation on an industrial scale. We have developed tobacco BY2 cell lines as an alternative plant-based platform for the production of cardosin B. These cultures successfully produced active cardosin B and a purification pipeline was developed to obtain isolated cardosin B. The enzyme displayed proteolytic activity towards milk caseins and milk clotting activity under standard cheese manufacturing conditions. We also identified an unprocessed form of cardosin B and further investigated its activation process. The use of protease-specific inhibitors suggested a possible role for a cysteine protease in cardosin B processing. Mass spectrometry analysis identified three cysteine proteases containing a granulin-domain as candidates for cardosin B processing. These findings suggest an interaction between these two groups of proteases and contribute to an understanding of the mechanisms behind the regulation and processing of plant APs. This work also paves the way for the use of tobacco BY2 cells as an alternative production system for active cardosins and represents an important advancement towards the industrial production of cardoon APs.

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“…This suggests that the same or closely related proteases account for bNAb processing in different plants irrespective of the type of tissue or cell line. Although the plant proteases responsible for cleavage within the CDR H3 loops of bNAbs have not yet been identified, previous inhibitor studies have indicated that serine and cysteine proteases could be involved in these processing reactions …”

Section: Discussionmentioning

confidence: 99%

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

Tarazona

Lobner

Taubenschmid

et al. 2019

Biotechnology Journal

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Broadly neutralizing antibodies (bNAbs) to human immunodeficiency virus type 1 (HIV‐1) hold great promise for immunoprophylaxis and the suppression of viremia in HIV‐positive individuals. Several studies have demonstrated that plants as Nicotiana benthamiana are suitable hosts for the generation of protective anti‐HIV‐1 antibodies. However, the production of the anti‐HIV‐1 bNAbs 2F5 and PG9 in N. benthamiana is associated with their processing by apoplastic proteases in the complementarity‐determining‐region (CDR) H3 loops of the heavy chains. Here, it is shown that apoplastic proteases can also cleave the CDR H3 loop of the bNAb 2G12 when the unusual domain exchange between its heavy chains is prevented by the replacement of Ile19 with Arg. It is demonstrated that CDR H3 proteolysis leads to a strong reduction of the antigen‐binding potencies of 2F5, PG9, and 2G12‐I19R. Inhibitor profiling experiments indicate that different subtilisin‐like serine proteases account for bNAb fragmentation in the apoplast. Differential scanning calorimetry experiments corroborate that the antigen‐binding domains of wild‐type 2G12 and 4E10 are more compact than those of proteolysis‐sensitive antibodies, thus shielding their CDR H3 regions from proteolytic attack. This suggests that the extent of proteolytic inactivation of bNAbs in plants is primarily dictated by the steric accessibility of their CDR H3 loops.

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Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production

Cited by 17 publications

References 50 publications

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

Expression of recombinant cardosin B in tobacco BY2 cells: an alternative system for the production of active milk clotting enzymes

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

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