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Recombinant production of plant lectins in microbial systems for biomedical application - the frutalin case study.

Oliveira C, Teixeira JA, Domingues L - Front Plant Sci (2014)

Bottom Line: The main problem of using such lectins as biomedical tools is that "batch-to-batch" variation in isoforms content may lead to inconstant results.The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit.Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

View Article: PubMed Central - PubMed

Affiliation: Centre of Biological Engineering, University of Minho Braga, Portugal.

ABSTRACT
Frutalin is a homotetrameric partly glycosylated α-D-galactose-binding lectin of biomedical interest from Artocarpus incisa (breadfruit) seeds, belonging to the jacalin-related lectins family. As other plant lectins, frutalin is a heterogeneous mixture of several isoforms possibly with distinct biological activities. The main problem of using such lectins as biomedical tools is that "batch-to-batch" variation in isoforms content may lead to inconstant results. The production of lectins by recombinant means has the advantage of obtaining high amounts of proteins with defined amino-acid sequences and more precise properties. In this mini review, we provide the strategies followed to produce two different forms of frutalin in two different microbial systems: Escherichia coli and Pichia pastoris. The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit. Emphasis is given particularly to recombinant frutalin produced in P. pastoris, which showed a remarkable capacity as biomarker of human prostate cancer and as apoptosis-inducer of cancer cells. Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

No MeSH data available.


Related in: MedlinePlus

Main strategies for the production of soluble recombinant FTL in Escherichia coli (A) and Pichia pastoris(B). Different frutalin codifying genes were cloned in E. coli and P. pastoris, which deduced amino-acid sequences share 93% of identity (Table 2 in Oliveira et al., 2009a).
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Figure 1: Main strategies for the production of soluble recombinant FTL in Escherichia coli (A) and Pichia pastoris(B). Different frutalin codifying genes were cloned in E. coli and P. pastoris, which deduced amino-acid sequences share 93% of identity (Table 2 in Oliveira et al., 2009a).

Mentions: A FTL cDNA sequence was used for production of recombinant FTL in E. coli by different strategies (Oliveira et al., 2009a). The first attempts to produce soluble EcrFTL in E. coli focused in the use of engineered E. coli strains that have extra copies of rare tRNAs and in the optimization of the induction conditions, but resulted in low yields (Oliveira et al., 2009a; Costa, 2013). The soluble production of EcrFTL from strain E. coli BL21 Codon Plus RIPL (DE3), harboring the pET-25b(+) expression vector (Novagen), was maximized to 16 mg/l by the implementation of an experimental factorial design (Oliveira et al., 2009a; Figure 1). However, all the experimental conditions resulted in EcrFTL produced predominantly as insoluble protein. Even though, EcrFTL was purified from crude E. coli extracts by sequential size exclusion (SEC) and cation ion exchange chromatography (IEC) that yielded 76 μg of protein per liter of E. coli culture. Purified EcrFTL migrated in SDS-PAGE gel as a homogeneous single-band protein with a molecular mass of about 17 kDa, indicating that the linker was not cleaved. Nevertheless, EcrFTL presented HA against rabbit erythrocytes, although it required more time to develop this activity than FTL. Thus, the HA of FTL is not strictly dependent on linker cleavage. In assays of HA inhibition by different sugars, EcrFTL presented specificity for galactose; however, it could not be purified by affinity chromatography on A. pavonina galactomannan, thus revealing lower sugar-binding affinity than FTL. The biomedical properties of this EcrFTL were not evaluated since the amounts obtained through this strategy were unsatisfactory and we were willing to improve them.


Recombinant production of plant lectins in microbial systems for biomedical application - the frutalin case study.

Oliveira C, Teixeira JA, Domingues L - Front Plant Sci (2014)

Main strategies for the production of soluble recombinant FTL in Escherichia coli (A) and Pichia pastoris(B). Different frutalin codifying genes were cloned in E. coli and P. pastoris, which deduced amino-acid sequences share 93% of identity (Table 2 in Oliveira et al., 2009a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126444&req=5

Figure 1: Main strategies for the production of soluble recombinant FTL in Escherichia coli (A) and Pichia pastoris(B). Different frutalin codifying genes were cloned in E. coli and P. pastoris, which deduced amino-acid sequences share 93% of identity (Table 2 in Oliveira et al., 2009a).
Mentions: A FTL cDNA sequence was used for production of recombinant FTL in E. coli by different strategies (Oliveira et al., 2009a). The first attempts to produce soluble EcrFTL in E. coli focused in the use of engineered E. coli strains that have extra copies of rare tRNAs and in the optimization of the induction conditions, but resulted in low yields (Oliveira et al., 2009a; Costa, 2013). The soluble production of EcrFTL from strain E. coli BL21 Codon Plus RIPL (DE3), harboring the pET-25b(+) expression vector (Novagen), was maximized to 16 mg/l by the implementation of an experimental factorial design (Oliveira et al., 2009a; Figure 1). However, all the experimental conditions resulted in EcrFTL produced predominantly as insoluble protein. Even though, EcrFTL was purified from crude E. coli extracts by sequential size exclusion (SEC) and cation ion exchange chromatography (IEC) that yielded 76 μg of protein per liter of E. coli culture. Purified EcrFTL migrated in SDS-PAGE gel as a homogeneous single-band protein with a molecular mass of about 17 kDa, indicating that the linker was not cleaved. Nevertheless, EcrFTL presented HA against rabbit erythrocytes, although it required more time to develop this activity than FTL. Thus, the HA of FTL is not strictly dependent on linker cleavage. In assays of HA inhibition by different sugars, EcrFTL presented specificity for galactose; however, it could not be purified by affinity chromatography on A. pavonina galactomannan, thus revealing lower sugar-binding affinity than FTL. The biomedical properties of this EcrFTL were not evaluated since the amounts obtained through this strategy were unsatisfactory and we were willing to improve them.

Bottom Line: The main problem of using such lectins as biomedical tools is that "batch-to-batch" variation in isoforms content may lead to inconstant results.The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit.Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

View Article: PubMed Central - PubMed

Affiliation: Centre of Biological Engineering, University of Minho Braga, Portugal.

ABSTRACT
Frutalin is a homotetrameric partly glycosylated α-D-galactose-binding lectin of biomedical interest from Artocarpus incisa (breadfruit) seeds, belonging to the jacalin-related lectins family. As other plant lectins, frutalin is a heterogeneous mixture of several isoforms possibly with distinct biological activities. The main problem of using such lectins as biomedical tools is that "batch-to-batch" variation in isoforms content may lead to inconstant results. The production of lectins by recombinant means has the advantage of obtaining high amounts of proteins with defined amino-acid sequences and more precise properties. In this mini review, we provide the strategies followed to produce two different forms of frutalin in two different microbial systems: Escherichia coli and Pichia pastoris. The processing and functional properties of the recombinant frutalin obtained from these hosts are compared to those of frutalin extracted from breadfruit. Emphasis is given particularly to recombinant frutalin produced in P. pastoris, which showed a remarkable capacity as biomarker of human prostate cancer and as apoptosis-inducer of cancer cells. Recombinant frutalin production opens perspectives for its development as a new tool in human medicine.

No MeSH data available.


Related in: MedlinePlus