Recombinant expression and biochemical characterization of sugarcane legumain

Santos-Silva, Ludier Kesser; Soares-Costa, Andréa; Gerald, Lee Tseng Sheng; Meneghin, Silvana P.; Henrique-Silva, Flávio

doi:10.1016/j.plaphy.2012.05.020

Cited by 15 publications

(7 citation statements)

References 59 publications

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“…This protein showed inhibitory activity against human cathepsins B, K, L, and V (Oliva et al 2004). CaneCPI-2 and CaneCPI-3 were characterized by Gianotti et al (2006) and were shown to inhibit papain; CaneCPI-3 also inhibits legumain (Santos-Silva et al 2012). CaneCPI-4 showed inhibitory activity against human cathepsins B and L (Gianotti et al 2008).…”

Section: Introductionmentioning

confidence: 99%

A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

et al. 2017

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Cystatin B was recently identified as an acid-resistant protein in acquired enamel pellicle; it could therefore be included in oral products to protect against caries and erosion. However, human recombinant cystatin is very expensive, and alternatives to its use are necessary. Phytocystatins are reversible inhibitors of cysteine peptidases that are found naturally in plants. In plants, they have several biological and physiological functions, such as the regulation of endogenous processes, defense against pathogens, and response to abiotic stress. Previous studies performed by our research group have reported high inhibitory activity and potential agricultural and medical applications of several sugarcane cystatins, including CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4. In the present study, we report the characterization of a novel sugarcane cystatin, named CaneCPI-5. This cystatin was efficiently expressed in Escherichia coli, and inhibitory assays demonstrated that it was a potent inhibitor of human cathepsins B, K, and L ( K = 6.87, 0.49, and 0.34 nM, respectively). The ability of CaneCPI-5 to bind to dental enamel was evaluated using atomic force microscopy. Its capacity to protect against initial enamel erosion was also tested in vitro via changes in surface hardness. CaneCPI-5 showed a very large force of interaction with enamel (e.g., compared with mucin and casein) and significantly reduced initial enamel erosion. These results suggest that the inclusion of CaneCPIs in dental products might confer protection against enamel erosion.

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Section: Introductionmentioning

confidence: 99%

A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

et al. 2017

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“…The recombinant cysteine protease was fused to the PHO1 signal peptide for secretion, but the protease was found trapped in the cell membrane. Nevertheless, several other cysteine proteases were successfully expressed and secreted in P. pastoris directed through the α-factor signal peptide [21][22][23]. Also our results suggest that the α-factor signal peptide efficiently secreted the recombinant propapain into the culture medium thereby facilitating protein purification.…”

Section: Heterologous Expression Of the Codon-optimized Propapain Seqmentioning

confidence: 78%

Expression of a Codon-Optimized Carica papaya Papain Sequence in the Methylotrophic Yeast Pichia pastoris

Hirth¹,

Zibek²

2015

J Microb Biochem Technol

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The cysteine endoprotease papain is one of the most widely used plant proteases for industrial applications. However the traditional isolation of papain from the latex of papaya plants cannot cover the world-wide? demand. To increase papain production for industrial applications, several expression systems were studied in the last years for its recombinant production. While expression in Eschericha coli resulted in accumulation of insoluble protein, expression in baculovirus/ insect system and Saccharomyes cerevisiae resulted in low yields of soluble protein inadequate for large-scale production. Here we describe the heterologous expression of a synthetic codon-optimized propapain sequence in the Pichia pastoris strains X33 (Mut +) and KM71H (Mut s). The recombinant propapain could be expressed as soluble protein and secreted in the culture medium through the α-factor signal peptide. Highest activities were obtained in the Mut s strain when cultivated in complex medium. After purification by Ni-NTA chromatography 463 mg/L recombinant propapain was obtained comparable to the so far highest reported propapain yields in E. coli after protein solublilization and refolding and with a specific activity similar to a commercial papain from papaya latex.

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“…Caution is necessary when using this grouping as there is no substantial sequence difference between the two types of legumains nor is the distinction enough to assign specific roles for each member of this family. This grouping also does not exclude their expression and activity in other tissues or developmental stages (Kinoshita et al., 1995; Gruis et al., 2004; Santos-Silva et al., 2012). In barley, it has been demonstrated that members of both the vegetative and seed types are almost ubiquitous and perform a multifunctional role in different organs (Julián et al., 2013).…”

Section: Classification and Genomicsmentioning

confidence: 99%

“…Although VPEs (C13 family) belong to the cysteine protease family, they share very little sequence similarity with other cysteine proteases and are also much less sensitive to inhibition by E64 (Müntz and Shutov, 2002). However, active VPEs are inhibited by a sub-group of cystatins containing a C-terminal extension (Martinez et al., 2007; Santos-Silva et al., 2012). In soybean, only one of the 19 cystatins has this domain (van Wyk et al., 2014); similarly, only one C-terminal extended cystatin has been identified in barley (Julián et al., 2013) and rice (Christoff et al., 2016).…”

Section: Protein Processing Activation and Activitymentioning

confidence: 99%

Plant Vacuolar Processing Enzymes

2019

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Plant proteomes contain hundreds of proteases divided into different families based on evolutionary and functional relationship. In particular, plant cysteine proteases of the C1 (papain-like) and C13 (legumain-like) families play key roles in many physiological processes. The legumain-like proteases, also called vacuolar processing enzymes (VPEs), perform a multifunctional role in different plant organs and during different stages of plant development and death. VPEs are similar to animal caspases, and although caspase activity was identified in plants almost 40 years ago, there still remains much research to be done to gain a complete understanding of their various roles and functions in plants. Here we not only summarize the current existing knowledge of plant VPEs, including recent developments in the field, but also highlight the future prospective areas to be investigated to obtain a more detailed understanding of the role of VPEs in plants.

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Recombinant expression and biochemical characterization of sugarcane legumain

Cited by 15 publications

References 59 publications

A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion

Expression of a Codon-Optimized Carica papaya Papain Sequence in the Methylotrophic Yeast Pichia pastoris

Plant Vacuolar Processing Enzymes

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