Secretory expression of thermostable alkaline protease from &lt;i&gt;Bacillus stearothermophilus&lt;/i&gt; FI by using native signal peptide and &lt;b&gt;α&lt;/b&gt;-factor secretion signal in &lt;i&gt;Pichia pastoris&lt;/i&gt;

Latiffi, Amaliawati Ahmad; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abdul; Oslan, Siti Nurbaya; Basri, Mahiran

doi:10.1266/ggs.88.85

Cited by 26 publications

(14 citation statements)

References 17 publications

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“…Similarly, recombinant human interferon-γ was not detected in P. pastoris X-33 when interferon cDNA with the native signal peptide was inserted into the expression vector downstream of the α-factor 43 . Production of thermostable alkaline protease from Bacillus stearothermophilus F1 in two different P. pastoris strains (GS115 and SMD1168H) was about 1.5-fold higher when the open reading frame was inserted into the expression vector without the native signal peptide 44 . This effect on maturation and secretion of the heterologous protein due to the presence of both native signal peptide and α-factor secretion signal, could also explain the longer induction time require for pPIC9-GALNS strains to reach the highest enzyme activity levels in comparison with that observed for pPIC9-nspGALNS strains.…”

Section: Discussionmentioning

confidence: 99%

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

Rodríguez-López

Alméciga-Díaz

Sánchez

et al. 2016

Sci Rep

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Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.

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Section: Discussionmentioning

confidence: 99%

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

Rodríguez-López

Alméciga-Díaz

Sánchez

et al. 2016

Sci Rep

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“…However, the effect of using a signal peptide from the native gene and α-factor secretion signal from S. cerevisiae in isolate SO needs to be investigated. The latest report has shown that an α-factor secretion signal from S. cerevisiae offered higher recombinant protein expression as compared to a native gene secretion signal in P. pastoris [27].…”

Section: Discussionmentioning

confidence: 99%

A newly isolated yeast as an expression host for recombinant lipase

Oslan

Salleh

Rahman

et al. 2015

Cellular and Molecular Biology Letters

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Pichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.

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“…However, the integrated plasmid of the present study was highly stable for many generations under nonselective condition. The same plasmid was designed to integrate into the yeast genome at the AOX promoter, but the plasmid lacked P. pastoris specific autonomous replication sequence (PARS) which is crucial for the yeast genome [23]. Preliminary screening of P. pastoris transformants by PCR showed that a few recombinants harboured the pPICZ α B/ α -amylase plasmid from GS115 strain which were subsequently used for protein expression.…”

Section: Resultsmentioning

confidence: 99%

Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

Gandhi

Salleh

Rahman

et al. 2015

BioMed Research International

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Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t 1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.

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Secretory expression of thermostable alkaline protease from <i>Bacillus stearothermophilus</i> FI by using native signal peptide and <b>α</b>-factor secretion signal in <i>Pichia pastoris</i>

Cited by 26 publications

References 17 publications

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

A newly isolated yeast as an expression host for recombinant lipase

Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia pastoris

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