Expression of glucose oxidase by using recombinant yeast

et al. 2003

Appl Environ Microbiol

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Saccharomyces cerevisiae was engineered to express different amount of heavy (H)-and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2 ؊ and ura3 ؊ for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H-and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 g per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Expression of Heteropolymeric Ferritin Improves Iron Storage inSaccharomyces cerevisiae

Kim¹,

Kim²,

et al. 2003

Appl Environ Microbiol

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“…Plasmids were maintained and propagated in E. coli HB101 or DH5␣ according to Sambrook et al (24). S. cerevisiae 2805 (MAT␣ pep4::HIS3 prb1 ⌬can1 GAL2 his3 ura3-52) was used as a recipient cell for ferritin expression (18).…”

Section: Methodsmentioning

confidence: 99%

Enhanced Iron Uptake ofSaccharomyces cerevisiaeby Heterologous Expression of a Tadpole Ferritin Gene

Shin

Kwon

et al. 2001

Appl Environ Microbiol

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We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 g per gram (dry cell weight) of the recombinant yeast but was 210 g per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.Iron is an essential trace element for most living organisms. However, its availability is limited by the low solubility of Fe(III) and the ability of intracellular free iron to cause the production of toxic radicals. Iron deficiency is a common and serious nutritional problem that afflicts an estimated 30% of the world's population (32), especially when vegetable-based diets are the primary food source (6,8). Animal feeds based primarily on cereals often lack iron. Once iron is introduced into a cell, an intracellular storage form of iron is required that is soluble, nontoxic, and bioavailable (11, 28).Ferritin (apoferritin-Fe complex) is an iron storage protein found in most living organisms (27). Genes encoding this protein have been isolated from various sources including humans, animals, amphibia, plants, fungi, and bacteria (28). Ferritin is a spherical macromolecule with a protein coat of 24 structurally equivalent subunits that can contain up to 4,500 iron atoms as a ferric oxyhydroxide polymer in its central core (29). The major role of ferritin is to provide iron for the synthesis of iron-containing proteins and to prevent damage from free radicals produced by iron-dioxygen interactions (29,30). In ferritin, there are two main subunits, heavy (also known as heart, or H) and light (as known as liver, or L). Tissue isoferritins have functional differences that may be related to the structural properties of the subunits (1, 26). The H-rich ferritins associate at a lower isoelectric point and lower iron content and accumulate and release iron relatively rapidly (1, 3). Moreover, it has been demonstrated that the Escherichia coli-expres...

“…To express different levels of LTB and LTB::ApxIIA#5, a low-copy-number integrative vector, YIplac128 (14), and a high-copy-number episomal vector, YEp352 (42), were engineered to express the LTB::ApxIIA#5 fusion proteins and LTB, respectively. We used the same promoter for the two vectors to minimize the discrepancy due to differences in promoter strength.…”

Section: Vector Constructionmentioning

confidence: 99%

Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

Park

et al. 2011

Clin Vaccine Immunol

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A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.