Improving the expression of mini-proinsulin in Pichia pastoris

País-Chanfrau, José M; García, Yuneski; Licor, Lisandra; Besada, Vladimir; Castellanos‐Serra, Lila; Cabello, Cecilia; Hernández, Lester; Mansur, Manuel; Plana, Liuba; Hidalgo, Abdel; Támbara, Yanet; Abrahantes-Pérez, María Del Carmen; Toro, Yoandris del; Valdés, Jorge Hernández; Martínez, Eduardo Justo

doi:10.1023/b:bile.0000044871.80632.f9

Cited by 17 publications

(6 citation statements)

References 11 publications

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“…It was suggested that limited proteolysis can be attributed to the fact that the solubility of the proteases decreases upon the addition of (NH 4 ) 2 SO 4 by sulfate conjugation, leading to decrease of the total proteolytic activity in the culture supernatant. País-Chanfrau et al ( 2004 ) also reported an increased expression of recombinant mini-proinsulin in P. pastoris in bioreactors, achieved by, among others, periodical addition of ammonium sulfate. In this study, we describe the successful expression of Tt CBH7 in P. pastoris , under high osmotic pressure and increased salinity conditions.…”

Section: Discussionmentioning

confidence: 97%

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

2018

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Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.

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Section: Discussionmentioning

confidence: 97%

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

2018

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“…The human mini-proinsulin is characterized by a C-chain peptide consisting of only 9 amino acids (21)(22)(23)(24), whereas the C-chain peptide of natural human proinsulin is made up of 35 amino acids. The bridge between B chain and A chain is only 16.12 Å, and 16 Å is the minimum distance between a five-AA protein; hence, a three to five amino acid should be able to connect the C terminal B chain and the N terminal A chain, forming a modified protein which has the same function as natural insulin.…”

Section: Discussionmentioning

confidence: 99%

Oral route of mini-proinsulin-expressing Ganoderma lucidum decreases blood glucose level in streptozocin-induced diabetic rats

Hu²,

Lee³

et al. 2007

Int J Mol Med

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Oral administration of insulin to control blood glucose has become a hot area of diabetes research. The major issue is to produce enough insulin and prevent insulin degradation in the acidic environment of the stomach. We hypothesize that the natural structure of the cell wall and the endoplasmic reticulum in Ganoderma lucidum should help resist the digestion of insulin produced in these cells, making this fungus a feasible production and a new delivery system for oral insulin. A new mini-proinsulin gene was constructed and was transformed into G. lucidum by the Agrobacteriummediated method. Driven by a strong promoter of GPD isolated from Lentinus edodes, expression of mini-proinsulin reached ≤10.4% of total soluble protein, equal to 174 μg/g wet weight, which is sufficient for oral route. Oral route of transgenic G. lucidum significantly reduced blood glucose in streptozocininduced diabetic rats, as compared to rats fed with saline (P<0.0002) or non-transgenic G. lucidum (P<0.0002). Blood glucose was reduced ≤64% in 80% of diabetic rats. Evaluation of pancreatic pathology showed that 54.5% rats in the transgenic group had no pathological changes, as compared to 25% in the saline group and 33.3% in the non-transgenic group. Rats in transgenic group had insulitis score <2, while 30% of rats in the saline group had insulitis score >2. These experimental results indicate that oral route of mini-proinsulinexpressing G. lucidum can be used to control blood glucose in diabetes and to improve pancreatic cell function.

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“…One of the main problems commonly encountered during recombinant protein production is protein loss due to protease degradation. We therefore modified the protocol suggested by Pai-Chanfrau and collegues [20] that evaluated the effect of temperature, pH and addition of excess nitrogen source and EDTA, on mini-pro-insulin expression. It was also previously observed that addition of casaminaocids reduced proteolysis of ovin interferon in P. pastoris by acting as preferential substrates [21].…”

Section: Discussionmentioning

confidence: 99%

“…Fermentation experiments were carried out in a Biostat C reactor (Sartorius Stedim; Melsungen, Germany) with initial working volume of 7 L. The medium used for all experiments modified from [20] contained: 20 g/L glycerol, 10 g/L casein hydrolysate, 13.4 g/L YNB, 0.0004 g/L biotin and 100 mM KH 2 PO 4 K 2 HPO 4 salts. Temperature was set at 22 °C, pH was fixed at 6.3 via automated addition of 30% v/v NH 4 OH and 30% v/v H 3 PO 4 .…”

Section: Methodsmentioning

confidence: 99%

Production of human pro-relaxin H2 in the yeast Pichia pastoris

et al. 2017

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BackgroundInitially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction.ResultsTo overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22 L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity.ConclusionsThis work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14 mg/L of product, as determined by densitometric analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0319-0) contains supplementary material, which is available to authorized users.

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Improving the expression of mini-proinsulin in Pichia pastoris

Cited by 17 publications

References 11 publications

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

Oral route of mini-proinsulin-expressing Ganoderma lucidum decreases blood glucose level in streptozocin-induced diabetic rats

Production of human pro-relaxin H2 in the yeast Pichia pastoris

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