Human sulfatase transiently and functionally active expressed in E. coli K12

Poutou-Piñales, Raúl A.; Niño, Adriana Vanegas; Landázuri, Patricia; Sáenz, Homero; Lareo, Leonardo; Peña, Olga Yaneth Echeverri; Avellaneda, Luis Alejandro Barrera

doi:10.2225/vol13-issue3-fulltext-8

Cited by 13 publications

(28 citation statements)

References 37 publications

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“…The results presented herein show that prokaryotic FGE can activate human sulfatases, reXecting high conservation of this mechanism and the possibility to produce active sulfatases in prokaryotic systems. These results agree with Wndings of active recombinant human IDS enzyme production in E. coli JM109 [21,36].…”

Section: Production Of Rgalns At Shake and Bench Scalessupporting

confidence: 90%

“…No band was recognized in samples from noninduced BL21/pGEX-GALNS and plasmid-free (BL21) strains, while rGALNS produced in CHO cells showed an expected 57-kDa band [50]. These results diVer from those of a recombinant iduronate-2-sulfate sulfatase (IDS) produced in Pichia pastoris [10,22] and E. coli JM109 [21,36], for which a large number of bands were recognized by an anti-IDS monoclonal antibody. The use of E. coli BL21 strain, which lacks the major protease, signiWcantly reducing endoproteolytic cleavage of damaged and recombinant proteins [45], could have a signiWcant eVect on the stability and processing of the produced rGALNS enzyme.…”

Section: Production Of Rgalns At Shake and Bench Scalesmentioning

confidence: 99%

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Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Rodríguez

Espejo

Hernández

et al. 2010

J Ind Microbiol Biotechnol

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Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

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Section: Production Of Rgalns At Shake and Bench Scalessupporting

confidence: 90%

Section: Production Of Rgalns At Shake and Bench Scalesmentioning

confidence: 99%

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Rodríguez

Espejo

Hernández

et al. 2010

J Ind Microbiol Biotechnol

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“…Later, recombinant IDS was produced in E. coli K12, DH5α, which allowed a significant improvement in the enzyme activity reaching up to 34.20 nmol h −1 mg −1 in the intracellular crude extract and a production of 94.3 ng IDS mL −1 [54]. Although these enzyme activities are lower than those reported for the purified enzymes produced in mammalian cells, it is important to highlight that under the same test conditions the IDS activity in serum and leukocytes samples from healthy donors was 0.22 ± 0.05 and 0.1 ± 0.02 nmol h −1 mg −1 , respectively [53]. This showed that active IDS might be produced in E. coli with enzyme activity levels similar or even higher than those observed in human samples.…”

Section: Recombinant Iduronate-2-sulfatasementioning

confidence: 76%

“…Active recombinant IDS was first produced in E. coli K12, JM109. At shake flask level, intracellular crude extract enzyme activity was 2.8 nmol min −1 mg −1 , while the extracellular enzyme activity was 1.2 nmol min −1 mg −1 [53]. Western-blot analysis of this recombinant IDS showed a band profile different to that expected for the human IDS, with four bands (62, 52, 49 and 40 kDa) for the intracellular enzyme, and five bands (97,49,43,41 and 40 kDa) for the extracellular enzyme [53,54].…”

Section: Recombinant Iduronate-2-sulfatasementioning

confidence: 92%

“…At shake flask level, intracellular crude extract enzyme activity was 2.8 nmol min −1 mg −1 , while the extracellular enzyme activity was 1.2 nmol min −1 mg −1 [53]. Western-blot analysis of this recombinant IDS showed a band profile different to that expected for the human IDS, with four bands (62, 52, 49 and 40 kDa) for the intracellular enzyme, and five bands (97,49,43,41 and 40 kDa) for the extracellular enzyme [53,54]. Later, recombinant IDS was produced in E. coli K12, DH5α, which allowed a significant improvement in the enzyme activity reaching up to 34.20 nmol h −1 mg −1 in the intracellular crude extract and a production of 94.3 ng IDS mL −1 [54].…”

Section: Recombinant Iduronate-2-sulfatasementioning

confidence: 92%

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Human recombinant lysosomal enzymes produced in microorganisms

Espejo-Mojica

Alméciga-Díaz

Rodríguez

et al. 2015

Molecular Genetics and Metabolism

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Production and characterization of a human lysosomal recombinant iduronate‐2‐sulfatase produced inPichia pastoris

Pimentel

Rodríguez-López

Diaz

et al. 2018

Biotech and App Biochem

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Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg H and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

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Human sulfatase transiently and functionally active expressed in E. coli K12

Cited by 13 publications

References 37 publications

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21

Human recombinant lysosomal enzymes produced in microorganisms

Production and characterization of a human lysosomal recombinant iduronate‐2‐sulfatase produced inPichia pastoris

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